Sheet-like composition

ABSTRACT

A sheet-shaped composition is provided which has an improved preservability and handling readiness, as well as a high flexibility in use. Amnion with trehalose added thereto is utilized. Addition of trehalose improves the flexibility of the amnion, and prevents basal membrane and stratum compactum from being damaged during lyophilization process.

This application is a continuation application of. U.S. application Ser.No. 11/989,296, filed Feb. 2, 2009, which is a 371 application ofPCT/JP2006/31425, filed Jul. 19,2006.

TECHNICAL FIELD

This invention relates to a sheet-shaped composition using an amnion anda method for producing the same. The sheet-shaped composition accordingto the invention is applicable as, for example, culture substrate forproducing artificial tissues (such as corneal epithelium), transplantmaterials for reconstructing eye surfaces, skins, etc, and asantiadhesive materials.

BACKGROUND ART

Amnion has preferable properties as transplant materials, such as highbiocompatibility and flexibility, and has found its use inreconstruction of corneal epithdlium and other various tissues (SeePatent Documents 1 through 4). Use of amnion can be largely classifiedinto two groups. Namely, it can be applied directly for any injured areato reconstruct the tissues, or can be used as a culture substrate forculturing cells. Flexibility of amnion is a crucial property for any oneof these applications. Because of its high flexibility, amnion can coverinjured site without any gaps, with a favorable adhesion and take ontothe injured site, resulting in a favorable therapeutic effect. On theother hand, when amnion is applied as a culture substrate for culturingcells, its high flexibility enables it to achieve a favorable cellamplification and normal organization (differentiation).

[Patent Document 1] Japanese Patent Publication No. 5-56987, the Gazette

[Patent Document 2] International Publication No. 03/043542, A1 Leaflet

[Patent Document 3] International Publication No. 03/92762, A1 Leaflet

[Patent Document 4] International Publication No.2004/078225, A1 Leaflet

DISCLOSURE OF THE INVENTION [Problems to be Solved by the Invention]

Amnion covers the outermost layer of uterine and placenta in mammals,and is derived during delivery. Accordingly, since fresh amnion cannotalways be available, long-term-preservation and readiness in handling ofamnion have been in demand for clinical application. Thus, derivedamnion has been stored in desiccated state for enhanced preservation andhandling readiness. The desiccation process, however, while providing asignificantly enhanced preservability, greatly reduces the flexibilityof the amnion after returning it back-to a wet state due to denaturedconstituent proteins. Such a low flexibility may impair coverage ofentire injured site, and lowers adhesiveness and take thereon. Inaddition, once desiccated, the amnion has a low cell proliferation ratethereon, and hampers the layering, thus causing no normal organization(differentiation). This may be due to drastically impaired flatness ofthe amnion surface accompanying the reduction of flexibility.

The present invention, seeking for the solution of the above problems,pursues to provide a sheet-shaped composition comprising amnion whichhas superior preservability and handling readiness, as well asflexibility in use.

[Means to Solve the Problems]

In order to achieve the above objective, the present inventors firsttried modifying amnion to enhance the flexibility. As a result, iftreated with trehalose, one of disaccharides, the amnion restored itsflexibility when returned .to its wet state, even if it had beendesiccated. Moreover, it was found that the restored flexibility was atan equivalent level to that of untreated amnions (raw amnion). Thus, itwas revealed that treatment with trehalose is effective in enhancing theflexibility of amnion.

Surprisingly, it was also shown that the treatment with trehalose had anadditional effect of enhancing transparency of amnion. Thus, it wasrevealed that treatment of the amnion with trehalose is extremelyeffective when amnion is used for any application where as high atransparency as possible is required (such as in cornealreconstruction).

In addition, since treatment with trehalose increased tensile strengthof the amnion and provided a tough amnion than raw ones, it was shownthat treatment with trehalose is effective in enhancing the handlingreadiness and preservability after transplant of the amnion.

Furthermore, since the biocompatibility of the amnion treated withtrehalose was determined to be as high as that of raw amnions, it wasdemonstrated that treatment with trehalose never adversely affects itsbiocompatibility.

With the knowledge thus accumulated, the effect of trehalose on thefunction of amniotic membrane as cell culture substrate was examined.Specifically, corneal epithelial cells were cultured on amnion that hadbeen treated with trehalose, and the cell proliferation rates andlayering thereon were determined. The results revealed a favorable cellproliferation and 5-7 layering, indicating a significant enhancementcomparative to those layering (1-2 layering) on amnion that had not beentreated with trehalose. Thus, it was shown that the treatment withtrehalose is effective also.in use of amnion as cell culture substrate.

Subsequently, a sheet of amnion with cell layers formed thereon wastransplanted on eye surface of animals to determine the reconstructioneffect. As a result, a favorable adhesion and take was revealed, with nodeficient in reconstructing the eye surface but with a high transparencyretained.

The present invention is mainly based on the above knowledge andprovides a sheet-shaped composition as described below.

[1] A sheet-shaped composition comprising an amnion with trehalose addedthereto.

[2] The sheet-shaped composition according to [1], in a frozen ordesiccated state.

[3] The sheet-shaped composition according to [2], in a lyophilizedstate.

The sheet-shaped composition according to [1] through [3], wherein theamnion is an amnion with epithelial cell layer removed.

[5] The sheet-shaped composition according to [1] through [4], whereinthe amnion has basal membrane components Collagen IV, Collagen VII, andLaminin 5 that are detected at an equivalent intensity to that inuntreated amnion.

[6] The sheet-shaped composition according to [1] through [5], whereinthe amnion is a human amnion.

[7] The sheet-shaped composition according to [1] through [6], whereincell layer consisting of tissue-derived cells is formed on the amnion.

[8] The sheet-shaped composition according to [7], wherein thetissue-derived cells are layered in the cell layer.

[9] The sheet-shaped composition according to [7], wherein thetissue-derived cells are derived from corneal epithelium, conjunctivalepithelium, skin epidermis, follicular epithelium, oral mucosaepithelium, pigment epithelium iris, pigment epithelium retina, airwaymucosa epithelium, or intestinal mucosa.

[10] The sheet-shaped composition according to [7], wherein the celllayer is composed of about 5-7 layered cells, and has properties similarto those of corneal epithelium.

[11] The sheet-shaped composition according to [1] through [6], for useas antiadhesive materials or reconstruction materials for surfacetissues damaged during surgical invasion.

[12] The sheet-shaped composition according to [1] through [11]; whereinthe amnion has any adhesive component attached on its chorion sidesurface.

[13] The sheet-shaped composition according to [12], wherein theadhesive component is fibrinogen and thrombin.

[14] The sheet-shaped composition according to [12], wherein theadhesive component is fibrinogen, thrombin and aprotinin.

[15] The sheet-shaped composition according to [1] through [14], whereinthe chorion side surface of the amnion is covered with bioabsorbablematerial.

In another embodiment, the present invention provides a transplantationmethod as follows.

[16] Transplant method using any one of sheet-shaped composition asimplant material.

In another embodiment, the present invention provides a method forproducing a sheet-shaped composition as follows.

[17] A method for producing a sheet-shaped composition, comprising thesteps of:

(a) preparing an amnion;(b) adding trehalose to said amnion.

[18] The method according to [17], further comprising the step of:

(c) freezing or desiccating said amnion after step (b).

[19] The method according to [18], further comprising the step of:

(d) sterilizing said amnion after step (c).

[20] The method according to any one of [17] through [19], wherein step(a) comprises the step of:

(a1) removing epithelium from said amnion.

[21] The method according to [20], wherein step (a1) comprises thefollowing steps of:

(1) preparing an amnion separated from an organism,(2) freeze-thawing said amnion,(3) subjecting said amnion after freeze-thawing to tryptic treatment,(4) washing said amnion after tryptic treatment.

[22] The method according to [21], wherein the freezing temperatureduring said freeze-thawing is from about −20° C. to about −80° C., andthe thawing temperature is from about 4° C. to about 50° C.

[23] The method according to [21] or [22], characterized by repetitionof said freeze-thawing process twice or more times.

[24] The method according to any one of [20] through [23], characterizedby the tryptic treatment being performed using a tryptic solution havinga tryptic concentration of from about 0.01% (w/v) to about 0.05% (w/v).

[25] The method according to [24], characterized by the tryptic solutioncomprising from about 0.1 mM to about 0.6 mM of a chelator selected fromthe group consisting of EDTA, NTA, DTPA, HEDTA, GLDA, and anycombination thereof.

[26] The method according to any one of [20] through [25], characterizedby the tryptic treatment being performed under the condition such thatthe tryptic solution is contacted with only the epithelium side of saidamnion.

[27] The method according to any one of [20] through [26], wherein thefollowing step of;

(A) forming a cell layer consisting of tissue-derived cells on saidamnion is performed prior to the step of (b).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a table showing the applications (applied sites, exemplaryapplication method, preferable form of the amnion, and main purpose) ofthe sheet-shaped composition as tissue-reconstructing material.

FIG. 2 is a series of graphs showing the procedure for securing amnions.The amnion in (a) is clamped between a pair of frame, and that in (b) isclamped between a frame and a plate-like member.

FIG. 3 is a graph showing the result of an evaluation test of physicalproperties (thickness) of the trehalose-treated and lyophilized amnion.

FIG. 4 is a graph showing the result of an evaluation test of physicalproperties (clarity) of the trehalose-treated and lyophilized amnion.

FIG. 5 is a graph showing the result of an evaluation test of physicalproperties (tensile strength) of the trehalose-treated and lyophilizedamnion.

FIG. 6 is a graph showing the result of an evaluation test of physicalproperties (flexibility) of the trehalose-treated and lyophilizedamnion.

FIG. 7 is a pair of photographs showing the result of an evaluation testof biocompatibility of the trehalose-treated and lyophilized amnion. Thetrehalose-treated and lyophilized amnion was transplanted to betweenrabbit corneal parenchyma layers, and the state of its eye surface wasmonitored. The left photograph in the left shows the state of eyesurface immediately after the transplant, and that in the right sideshows the state of eye surface 1 month after the transplant.

FIG. 8 is a photograph showing the result of an evaluation test ofbiocompatibility of the trehalose-treated and lyophilized amnion. Thetrehalose-treated and lyophilized amnion was transplanted to betweenrabbit corneal parenchyma layers, and a part of the cornea including thetransplanted region was isolated and subjected to HE staining at 1 monthafter the transplant.

FIG. 9 is a sectional view schematically showing the state ofinstruments for culturing the corneal epithelial cells on thetrehalose-treated lyophilized amnion. Culture insert 12 was stood stillon culture dish 11, on the bottom surface of which is formed 3T3 celllayer 15. On the other hand, an amnion 13 was stood still on the bottomsurface of the culture insert 12, to receive and culture cornealepithelial cells 14 thereon. Numeral 16 denotes the culture media.

FIG. 10 is a pair of photographs (HE staining image) showing the celllayer formed on the trehalose-treated and lyophilized amnion. Forcomparison, the cell layer formed on the amnion after lyophilization butwithout the trehalose-treatment (Un-treated lyophilized amnion) isshown.

FIG. 11 is a series of photographs showing HE staining images andimmuno-staining images of the sheet (cultured corneal epithelial sheet)with the cell layer formed on the trehalose-treated and lyophilizedamnion. Signals for each antibody in the immuno-staining images arecolored green. Cell nuclei are colored red. Corneal keratin is expressed(+), with the keratinized skin-type of keratin 10 (−) and corneal typeof keratin 13 (−) not expressed.

FIG. 12 is a series of photographs showing the reconstruction effect ofthe cultured corneal epithelial sheet made of the trehalose-treated andlyophilized amnion. The states of eye surface 2 days and 14 days afterthe cultured corneal epithelial sheet transplant (upper) and theirfluorescein staining images (lower) are shown.

FIG. 13 is a series of photographs showing the HE staining images (upperleft) and immuno-staining images (upper right and lower) for variouskeratins of the cultured corneal epithelial sheet 2 weeks after thetransplant. Mark ** denotes trehalose-treated and lyophilized amnions(TH-AM).

FIG. 14 is a series of photographs showing the results ofimmuno-staining of the trehalose-treated and lyophilized amnions usingan antibody directed against a basal membrane specific component and anantibody directed against a stratum compactum specific component. Theimmuno-staining images (C) of the trehalose-treated and lyophilizedamnion are shown. in comparison to those of raw amnions (A) and oflyophilized amnion without the trehalose-treatment (B). 1; Stainingimages of Collagen I, 2; Staining images of Collagen III, 3; Stainingimages of Collagen IV, 4; Staining images of Collagen V, 5; Stainingimages of Collagen VII, 6; Staining images of laminin 5, and 7; Stainingimages of Fibronectin.

FIG. 15 is a flow chart showing the removal procedure of amnioticepithelium.

FIG. 16 is a pair of figures showing the procedure of tryptic treatment.In the figure (a), the amnion secured in a frame is immersed in trypticsolution with its epithelial side down. In the figure (b), trypsin isacted upon the epithelial side of the amnion by pouring tryptic solutioninto the frame.

FIG. 17 is a series of photographs showing HE staining images oftrypsin-treated, raw (with epithelium attached), and manually-treatedamnions.

FIG. 18 is a series of photographs showing the immuno-staining images ofthe trypsin-treated amnion.

FIG. 19 is a series of photographs showing the immuno-staining images ofthe trypsin-treated amnion.

FIG. 20 is a series of photographs showing the immuno-staining images ofthe raw (with epithelium attached) amnion.

FIG. 21 is a series of photographs showing the immuno-staining images ofthe manually-treated amnion.

FIG. 22 is a table summarizing the results of HE staining experiment andimmuno-staining experiment.

EXPLANATION OF THE NUMERALS

1, 2, 3 frame

4 plate-like member

5 tryptic solution

10 amnion

11 culture dish (culture dish)

12 culture insert (culture insert container)

13 amnion

14 corneal epithelial cells

13 3T3 cell layer

16 culture medium

The Best Mode of Carrying Out the Invention

In one embodiment, the present invention relates to a sheet-shapedcomposition. According to the invention, the sheet-shaped compositionutilizes an amnion as its main component. Because of its high clarityand strength, amnion can form sheet-shaped composition with an improvedclarity and strength. Moreover, a high biocompatibility and lowimmunogenicity of amnion lead to an improved biocompatibility and lowimmunogenicity of the resulting sheet-shaped composition. Use of amnioncan be expected to exert various actions, such as anti-inflammatoryaction, suppression of scar formation, and inhibition of angiogenesis.Use of amnion is preferable also in respect of favorable formation ofthe cell layer, if contained in the sheet-shaped composition accordingto the invention. Specifically, as will be described later, if thesheet-shaped composition comprising the cell layer is formed by seedingand culturing a predetermined type of cells on an amnion which serves assubstrate (support), such a use of amnion results in a favorableadhesion and proliferation of cells, as well as a formation of the celllayers since an amnion has a property of allowing a favorable adhesionand proliferation of cells thereon.

(Origin of Amnion)

“Amnion” is a membrane covering the outermost layer of uterine andplacenta in mammals, and consists of an underlying collagen-richparenchyma, basal membrane thereon and epithelial layer. Amnion of, forexample, human, monkey, chimpanzee, swine, horse and bovine can be used.Amongst them, human amnion is preferably used because its safety isreliable, including its low immunogenicity and virusinfection-probability.

[Addition of Trehalose]

The sheet-shaped composition according to the present invention utilizesan amnion with trehalose added thereto. The inventors have found thatthe addition of trehalose improves the flexibility of the amnion,especially when the amnion is lyophilized. In addition, as will be shownin the Embodiments described later, it was found that the amnion withtrehalose added thereto serves favorably as a substrate for culturingcells. The sheet-shaped composition according to the invention that isconstructed based on such knowledge has a high flexibility, and allowsfor favorable proliferation and layering of cells when used as asubstrate for culturing cells.

One might focus on degradation of matrix proteins contained in anamnion, which leads to its lower strength, making the amnion moresusceptible to damages. Moreover, the amnion that has its matrix proteindegraded can no longer hold its moisture tight inside, become brittleand less resilient. The trehalose added to the amnion is expected to actupon the site of the matrix proteins where binding has loosened, therebyreinforcing the binding between proteins, normalizing the moisturecapture inside the amnion, and maintaining the moisture, integrity andflexibility native to the amnion. Further, by adding trehalose, thematrix proteins contained in the amnion may be prevented from becomingsoft during lyophilization process, and can effectively be protectedfrom swelling and weakening in water.

Trehalose (material name, general name) is a compound the is representedby α-D-Glucopyranosyl (1,1)-α-D-Glucopyranoside.

Addition of trehalose to an amnion can be performed by, for example,treating the amnion with trehalose solution. Exemplary method of addingtrehalose will be described later in detail.

In one embodiment of the invention, the composition consists essentiallyof an amnion with trehalose added thereto.

(State of the Amnion)

In one embodiment of the invention, an amnion having its epithelial celllayer removed is utilized. An amnion with its epithelial cell layerremoved is extremely safer since it does not cause any immunologicalrejection or other problems arising against epithelial cells. Inaddition, since cell adhesion and proliferation that can take place onthe amnion with its epithelium removed produce superior results, aquality cell sheet can be constructed in a shorter time period, thusproviding an advantage in the manufacture of the sheet-shapedcomposition.

Absence of the epithelial layer on the amnion can be confirmed bydetermining an absence of any cells of the amniotic epithelial layer onthe resultant sheet-shaped composition according to the invention.

On the other hand, an amnion with its epithelial layer retained can beutilized in constructing the sheet-shaped composition according to theinvention. Retention of the epithelial layer on the amnion allows one toperform a thorough sterilization procedure such as γ-ray treatment,thereby ensuring the safety of the sheet-shaped composition.

(Use of Reconstructed Amnion)

A reconstructed amnion can be used to construct the sheet-shapedcomposition according to the invention. Specifically, an amnion can besubjected to homogenizer, ultrasound, enzymatic or other treatment todecompose it, and then reconstructed into a membrane-like form.Treatment may be preferably performed using a homogenizer since it isexpected to relatively highly retain a structure having a minute basalmembrane. Treatment using a homogenizer may be performed at (arevolution speed of) 3000 rpm through 50000 rpm, preferably 10000 rpmthrough 40000 rpm, more preferably at about 30000 rpm.

(Thickness)

Use of an amnion for the sheet-shaped composition according to theinvention enables to attain an extremely thin sheet. The sheet-shapedcomposition according to the invention can be prepared to be as thin as,for example, 10 μm through 500 μm. Such an extreme thinness of the sheetallows for universal use of the composition. An amnion with a part (forexample, approximately 10 μm through 30 μm) of the stratum compactum ofon the chorionic membrane side removed may be used to construct thesheet-shaped composition according to the invention. Alternatively, anybioabsorbable material may be coated to attain the thickness of, forexample, 100 μm through 500 μm.

(Use of Adhesive Component)

In one embodiment of the present invention, on the surface of amnion,fibrinogen and thrombin (hereinafter, which are also collectivelyreferred to as “adhesive components”) is attached. Thus, when thesheet-shaped composition of the present invention is transplanted,firstly, fibrinogen is specifically hydrolyzed by thrombin so as to formfibrin, and then, fibrins are polymerized so as to form a stable fibrinclot which exhibits an adhesive effect. By virtue of the highadhesiveness, the sheet-shaped composition, once attached on a lesion,can attain a sufficient adhesion without suture, thus facilitating thesurgical procedure. In the present specification, an amnion that has anepithelium and with any adhesive components attached thereto may bereferred to as “amnion with attached adhesive components and epithelium”and that without an epithelium but having adhesive components attachedthereto as “amnion with attached adhesive components but withoutepithelium”.

Fibrinogen and thrombin are attached on either or both sides of theamnion depending on the application of the sheet-shaped composition ofthe present invention. In the case of one-sided attachment, thechorionic side-surface of the amnion (i.e. the surface opposite to itsepithelium) receives the attachment, regardless of a presence or absenceof the epithelium of the amnion. Accordingly, when in use, such asheet-shaped composition thus constructed is transplanted to theapplication site with the side that had its epithelium facing upward.Similarly, a sheet-shaped composition for use as anti-adhesive receivesits adhesive components on an either side of the amnion. On the otherhand, a sheet-shaped composition that is transplanted in vivo asbioadhesive appropriately receives adhesive components on both sides ofthe amnion.

As mentioned below, the sheet-shaped composition of this embodiment isprepared in an appropriate state (for example, dry state or wet state)through a step of attaching fibrinogen and thrombin to the surface ofamnion by considering the intended uses. Therefore, fibrin is expectedto be generated from a part of fibrinogen before the sheet-shapedcomposition is used depending upon the state during process and/or thefinal state. Therefore, the sheet-shaped composition of the presentinvention may include fibrin or a fibrin clot generated by such areason.

The origin of the fibrinogen and thrombin is not particularly limited.The fibrinogen and thrombin can be prepared by using blood of, forexample, human, monkey, chimpanzee, bovine, horse, sheep, pig, and thelike, as a starting material. Furthermore, as the fibrinogen andthrombin, a recombinant obtained by using cultured cells (for example,CHO cells or COS cells) may be used. It is preferable to use fibrinogenand thrombin derived from human (in particular, human-derivedrecombinant). This is advantageous from the viewpoint of safetyincluding immunogenicity. Furthermore, by considering the stable qualityand problem of infection, it is particularly preferable to use arecombinant.

It is particularly preferable to use fibrinogen and thrombin derivedfrom blood of a patient (recipient) who is going to be subjected totransplantation of the sheet-shaped composition of the presentinvention. This is advantageous because immunological rejection may notbe induced.

Note here that the origins of the fibrinogen and thrombin may notnecessarily be the same. For example, the combination of fibrinogenderived from human blood and thrombin derived from bovine blood may beused.

The attached amount of fibrinogen and thrombin is not particularlylimited. For example, the attached amount of fibrinogen can be set inthe range from 0.1 mg to 50 mg per 1 cm²of amnion. Similarly, theattached amount of thrombin can be set in the range from 0.5 μm to 10 mgper 1 cm²of amnion.

Adhesive force is primarily considered when setting the attached amountof fibrinogen and thrombin. That is to say, in order to obtain thenecessary adhesion force, the attached amounts of these components needto be set. On the other hand, when the attached amount of fibrinogen andthrombin is too large, immune reaction or angiogenesis may tend to beinduced, although depending upon the origin of fibrinogen to be used.

Herein, in a case that a sheet-shaped composition applied toreconstruction of the ocular surface (for example, when angiogenesis dueto these components after transplantation may occur) in order tosuppress the induction of the angiogenesis as much as possible, it ispreferable that the attached amount of these components is reduced. Bysetting the attached amount of these components as small as possible,the angiogenesis after transplantation can be suppressed and hightherapeutic effect can be expected. Ai described in the below mentionedexample, as a result of the investigation by the present inventors, whenthe attached amount of fibrinogen is about 0.5 mg or more per 1 cm² ofamnion, the excellent adhesive force with respect to the ocular surfacewas observed. As to thrombin, even when the attached amount of thrombinis 1 μg per 1 cm² of amnion, the excellent adhesive force with respectto the ocular surface was observed. Based on these findings, thepreferable range of the attached amount of fibrinogen is 0.5 mg to 20 mgper 1 cm² of amnion. Further preferable range is 0.5 mg to 10 mg per 1cm² of amnion. More preferable range is 0.5 mg to 6 mg (specifically,for example, about 0.5 mg, about 1 mg, and about 2 mg) per 1 cm² ofamnion. Similarly, the preferable range of the attached amount ofthrombin is 1 μg to 1 mg per 1 cm² of amnion. Further preferable rangeis 5 μg to 200 μg per 1 cm² of amnion. More preferable range is 10 μg to100 μg (specifically, for example, about 10 μg, about 20 μg, and about30 μg) per 1 cm² of amnion.

In one embodiment of the present invention, in addition to fibrinogenand thrombin, aprotinin is attached to the surface of amnion. Aprotinininhibits the fibrin clot formed by the effect of thrombin from beingdissolved by plasmin. Therefore, by using aprotinin together, thedecomposition of the fibrin clot can be suppressed. As a result, theadhesive force can be maintained or reinforced.

The origin of aprotinin is not particularly limited. The aprotininderived from the pancreas of, for example, bovine, horse, sheep, pig,monkey, chimpanzee, and the like. Furthermore, a recombinant aprotininobtained by using cultured cells (for example, CHO cells or COS cells)may be used. It is preferable to use a recombinant from the viewpoint ofthe stable quality and problem of infection.

When aprotinin is used, the attached amount thereof is not particularlylimited. For example, the attached amount of aprotinin can be set in therange from 0.1 KIU to 200 KIU per 1 cm² of amnion. We examined how achange in the attached amount of aprotinin affects the adhesion force inaddition to the investigation of the above-mentioned attached amount offibrinogen. As a result, even when the amount of aprotinin is set in therange from 1 KIU to 2 KIU, a sufficient adhesive force to the ocularsurface was observed. Based on this finding, the preferable range of theattached amount of aprotinin is 1 KIU to 100 KIU per 1 cm² of amnion.Further preferable range is 1 KIU to 20 KIU per 1 cm² of amnion. Morepreferable range is 1 KIU to 10 KIU (specifically, for example, about 1KIU, about 2 KIU, and about 3 KIU) per 1 cm² of amnion. When the amountof the aprotinin is too large, the manufacturing cost is increased andfurthermore, the side effect caused by the immunogenicity, etc. of theaprotinin itself may be increased. On the other hand, when the amount ofaprotinin is too small, the effect of aprotinin of suppressing thedeposition of fibrin clot may not be exhibited sufficiently.

In various purposes, the fibrin clot is used as an adhesive, generallywith aprotinin. As a result of the investigation by the presentinventors, in the sheet-shaped composition of the present invention,even if aprotinin is not used, it has been found that sufficientadhesive force with respect to a living body can be obtained. Whenaprotinin may not be used, a configuration can be simplified, so thatadvantages in terms of manufacture and cost can be achieved. Inaddition, it becomes unnecessary to consider the side effect caused bythe immunogenicity etc. of the aprotinin itself.

(Reinforcement by Bioabsorbable Material)

Chorionic side of the amnion can be covered with any bioabsorbablematerial to reinforce the sheet-shaped composition of the invention.Bioabsorbable materials for such a purpose are preferably any materialthat is degraded and absorbed earlier relative to the amnion. Forexample, polygractin 910, gelatin, collagen and polylactic acid may bepreferably used as bioabsorbable material described herein. The shape ofthe bioabsorbable material for reinforcement is not limited.Biomaterials formed into, for example, a mesh or sheet may be used tocover the chorionic side of the amnion to reinforce the amnion. Theamnion during the reinforcement process may be either of a moistenedstate or desiccated state, although the amnion in the. final product ispreferably in a desiccated state in which the amnion is superior inrespect of handling readiness and storage. The amnion with suchreinforcement is referred to in the present specification as “hybridamnion”.

(Cell Layer)

In another embodiment of the present invention, the sheet-shapedcomposition comprises a cell layer on the amnion. If any adhesivecomponents are utilized in such a form of composition, those adhesivecomponents (such as fibrinogen) are attached to the side of the amnionwhere no cell layer is formed.

In this embodiment, amnion from which the epithelium has been removed isgenerally used. Then, at the side where the epithelium has been present,a cell layer is formed. This cell layer is formed from cells ofbiological origin. The origin of the cells constituting the cell layeris not particularly limited. Examples of the cells include cells derivedfrom corneal epithelium, conjunctival epithelium, skin epidermis, hairfollicle epithelium, oral mucosal epithelium, iris pigment epithelium,retina pigment epithelium, respiratory tract mucosa epithelium orintestinal tract mucosa epithelium, and the like. A cell layer may beformed by using two types or more of cells that are different from eachother. Formation from two types or more of cells that are derived fromdifferent origins is also referred to as “hybridization” in thisspecification. The form in which cells are contained in the hybridizedcell layer (state of hybridization) is not particularly limited and, forexample, cells may be dispersed or some cells (or plural types of cells)may be present as a group. Furthermore, the content of cells may not beuniform over the entire cell layer. The cell layer may be a single layeror multilayer (stratified layers).

The type of cells forming the cell layer, if any, on the amnion will nowbe described hereinafter, taking an example of a sheet-shapedcomposition for reconstruction of corneal epithelium.

The hybridized cell layer contains two types or more of cells. One typeof cells is referred to as first cells and the other type of cells thatare different from the first cells are referred to as second cells forconvenience of explanation. Firstly, autologous cells are used as thefirst cells. In this specification, “autologous” indicates a subject towhom the sheet-shaped composition of the present invention is to beapplied, that is, a subject who undergoes transplantation (recipient).On the other hand, other than such “autologous” is referred to as“allogeneic.” The type of the first cells is not particularly limited aslong as the first cells can form a corneal epithelium-like mucosalepithelium layer when they are hybridized with the below-mentionedsecond cells. Examples of the first cells include cells derived frommucosal epithelium such as oral mucosal epithelium, conjunctivalepithelium, and nasal mucosal epithelium, or cells derived fromundifferentiated cells capable of constructing such mucosal epithelium(that is, mucosal epithelium stem cells). Herein, the term “derived fromor of origin” is used for the purpose of specifying a starting material.Therefore, for example, cells derived from (of origin of) the oralmucosal epithelium indicates cells obtained by using oral mucosalepithelial cells as a starting material. Furthermore, in the presentinvention, the term “undifferentiated cells capable of constructing suchmucosal epithelium” indicates cells having the potency ofdifferentiating into cells constituting mucosal epithelium. For example,undifferentiated cells capable of constructing oral mucosal epitheliumindicates cells capable of differentiating into oral mucosal epithelialcells. Specific examples of the undifferentiated cell include aprecursor cell or a stem cell of cells constituting specific tissue, forexample, oral mucosal epithelium or conjunctival epithelium, and thelike, or an epithelial stem cell with lower differentiation.

The hybridized cell layer may include two or more different types of thefirst cells. For example, a cell layer may be constructed from cellsderived from oral mucosal epithelium and cells derived from conjunctivalepithelium.

The “oral mucosal epithelium” in the present invention may include oralcrevicular mucosal epithelial part, labial part, palate part, buccalpart, and the like. Whether or not the cells are derived from oralmucosal epithelium can be confirmed by using, as an indicator, theexpression of keratin 4 or keratin 13 specific to oral mucosalepithelium. Alternatively, the expression of keratin 3 can be used as anindicator. This keratin 3 is known to be one of the cornea-specifickeratins. However, keratin 3 was reported to be expressed also in theoral mucosal epithelial cell. Note here that it is preferable that oralmucosal epithelial cells are used as a material for producing acomposition for transplantation of corneal epithelium from a viewpointthat it expresses this cornea-specific keratin, keratin 3.

On the other hand, by examining the expression of genes specific to anoral mucosal epithelial cell, it can be confirmed that cells are derivedfrom oral mucosal epithelium.

Similarly, in the case of cells derived from a tissue other than oralmucosal epithelium, by examining the expression of the marker or genespecific to the tissue, the origin thereof can be confirmed.

Specific examples of the second cells include cells derived from cornealepithelium, conjunctival epithelium or amnion epithelium. Among them, itis preferable that the second cells are cells derived from cornealepithelium or conjunctival epithelium. This is advantageous because thecell layer constructed by cells derived from ocular surface tissue canhave a property closer to that of corneal epithelium. It is particularlypreferable that the second cells are derived from corneal epithelium.This is advantageous because a layer that is more similar to cornealepithelium can be obtained.

The second cells may be autologous cells or allogeneic cells. Whenautologous cells are used, a cell layer with no or little problem ofimmunological rejection can be obtained. When allogeneic cells are used,since it is easy to prepare cells, it is advantageous from the viewpointof manufacturing. The cell layer of the present invention may includetwo or more different types of second cells. For example, the cell layermay be constructed in a state which includes, for example, cells derivedfrom corneal epithelium and cells derived from conjunctival epithelium.

Whether or not the cell layer in the sheet-shaped composition of thepresent invention includes cells that are derived from the cornealepithelium can be confirmed by using, as an indicator, the expression ofkeratin 3 or keratin 12 specific to corneal epithelium. Alternatively,the expression of keratin 4 can be used as an indicator.

Similarly, in the case of cells derived from the tissue other thancorneal epithelium, by examining the expression of marker or genespecific to the tissue, the origin thereof can be confirmed.

Since the sheet-shaped composition of the present invention employsamnion as a support, it can be constructed very thinly. When thesheet-shaped composition of the present invention does not include acell layer, the sheet-shaped composition can be prepared to thethickness, for example, in the range from 10 μm to 100 μm. When thesheet-shaped composition includes a cell layer, it can be prepared tothe thickness, for example, in the range from 20 μm to 200 μm. Thus,very thin state is also one of the main features of the presentinvention. With this feature, the composition becomes applicable to awide spectrum of uses. In particular, making the most of the hightransparency, it can be applied to reconstruction of the ocular surface.

(State of the Sheet-Shaped Composition)

The state of the sheet-shaped composition according to the invention isnot limited, and may be presented either in a moistened state (by, forexample, immersing in any solution), in a frozen state, or in adesiccated state (including semi-desiccated state). Frozen or desiccatedstate is advantageous in respect of handling readiness and storage. Whendesiccated, the composition can be stored at a normal temperature (forexample about 10° C. through about 35° C.). Specifically, the storage ina freezer or refrigerator until use is no longer required, and thecomposition will be more readily handled (storage, transportation,etc.). Of course, the composition in a desiccated state may be stored ina freezer or refrigerator, if need be.

Above all, lyophilized state provides a handling readiness and favorableattachment to the lesion where it is applied (where it filtrates andexerts its adhesiveness), thereby makes a suture after the applicationunnecessary (Of, course, suture may be performed to ensure attachment tothe lesion). Abrogation of suture greatly reduces the burdens onpatients and physicians.

Preferably, the sheet-shaped composition according to the invention isconstructed utilizing an amnion, with an epithelial layer in a frozenstate, with an epithelial layer in a desiccated state (lyophilizedstate), without an epithelial layer in a moistened state, or without anepithelial layer in a desiccated state (lyophilized state). In thepresent specification, an amnion with an epithelial layer in a frozenstate is also referred to as “freezing-stored amnion with anepithelium”, that with an epithelial layer in a lyophilized state as“lyophilized amnion with an epithelium”, and that without an epitheliallayer in a lyophilized state as “lyophilized amnion without anepithelium”.

The sheet-shaped composition in a desiccated state is ready to handle,and, specifically, can be stored even at a normal temperature (forexample about 10° C. through about 35° C.). Thus, storage in a freezeror refrigerator until use is no longer necessary, thus making itshandling (such as storage or transportation) ready. Moreover,desiccation makes it possible to effectively sterilize the amnionwithout affecting its use. Further, since degradation of the amnion in adesiccated state is extremely low, its high quality can be maintainedfor a longer period of time.

It is believed that retention of the structure of basal membrane iscrucial for the sheet-shaped composition of the invention to perform thefunction expected for it (such as a function as a substrate to form acell layer and as a tissue-reconstruction material). In a preferableembodiment of the present invention, an amnion with its basal membranecomponents (Collagen IV (α1, α2, and α5), Collagen VII, laminin 5)retained is utilized. Presence or absence of the basal membranecomponents can be assayed by performing an immuno-staining targeting thecomponents at issue for detection. In one embodiment of the invention,at least one, preferably a plurality of these components, or even all ofthese components are detected. It is also preferable for thesecomponents to be detected at intensity not significantly different fromthose detected for an untreated amnion (i.e. amnion which has undergonetreatment, such as freezing, post isolation from an organism).

In addition, an amnion with components of stratum compactum (Collagen I,III, V and Fibronectin) retained may preferably utilized. The retainedstate of the stratum compactum components may be detected animmuno-staining, similarly to the detection of the basal membranecomponents described above.

By adding trehalose to the amnion, the structures of the basal membraneand stratum compactum are highly maintained, even after lyophilizationprocess.

(Presented Form)

The sheet-shaped composition according to the invention may be presentedin any container, such as glass of plastic-made container, or in apackaged form in a clear film or sun-blocking sheet.

Preferably, the sheet-shaped composition of the invention is presentedin a packaged form so that it has substantially no contact with oxygen.Under such a condition, the quality of the composition will cause nodeterioration due to oxygen, but will be maintain at a high level for alonger period of time. A condition that “has substantially no contactwith oxygen” will be attained through use of a container evacuated orfilled with nitrogen gas (i.e. exhausted with nitrogen), or in the formof air-tight package using a film or sheet. In any case, thesheet-shaped composition of the invention is typically sterilized priorto use.

(Application)

The sheet-shaped composition according to the invention can be appliedas, for example, transplant material for tissue reconstruction, andanti-adhesive material. Medical fields where the sheet-shapedcomposition of the invention can be applied include ophthalmology,digestive surgery, gynecology and dermatology.

Exemplary applications (application sites and methods, for example) ofthe sheet-shaped composition according to the invention will now bedescribed with respect of situations (A) where the sheet-shapedcomposition of the invention comprises no cell layer and those (B) whereit comprises a cell layer.

A. Situations where the sheet-shaped composition comprises no celllayer.

The sheet-shaped composition with no cell layer can be applied for usein, for example, reconstruction of sclera and cornea (treatment ofpterygium and corneal epithelial defects), and coverage of skin(epidermis) ulcer and burn. It is also applicable as tissuereconstruction material. The term “tissue reconstruction material”herein refers to any material that can be used for reconstruction(regeneration) of any tissue of an organism. The sheet-shapedcomposition with h no cell layer can be favorably used in a therapy forreconstructing surface tissue of organ or apparatus damaged duringsurgical invasion. The sheet-shaped composition of the invention ispreferable particularly for reconstructing surface tissue that wouldcause adhesion during a normal healing process. The term “tissuereconstruction” herein typically refers to recovery of damaged lesion ofsurface tissue into a normal state. Alternatively, the term may compriserecovery of an organ or apparatus into a normal state by preventingre-adhesion of surface tissue of it (for example, preventing a salpinxfrom re-adhesion post adhesiotomy to return it to a normal state).

Exemplary tissues for reconstruction according to the invention includesurface tissues of abdominal, thoracic or intrapelvic organ or apparatus(such as stomach, colon, small intestine, blind intestine, duodenum,heart, lung, oviduct, intestinum rectum, liver, ovarium, uterine), orsurface tissues of intraperitoneal, intrathoracic, intrapelvic, oral,nasal, ear, or throat cavity, or ocular tissues. Accordingly, thesheet-shaped composition of the invention can be utilized in the fieldof digestive surgery, obstetrics and gynecology, thoracic surgery, oralsurgery, ear, nose and throat surgery, and ophthalmic surgery. Thesheet-shaped composition of the invention is particularly suitable as amaterial for reconstructing surface tissues of abdominal, thoracic orintrapelvic organ or apparatus, or abdominal cavity, thoracic cavity orsurface tissues. On the other hand, the present invention is alsoapplicable to the other fields which accompany surgical operation.Details of the sites, method, etc., of applying the sheet-shapedcomposition of the invention will now be described.

Use of the sheet-shaped composition of the invention applied as a tissuereconstruction material can be generally classified according to theapplication method and application purpose into the following threeclass.

(1) The Coverage (Application Method) as a Tissue ReconstructionMaterial/Tissue Reconstruction (Application Purpose)

Use (usage) of the composition for the purpose of reconstructing thedamaged tissue surface by applying the tissue reconstruction materialonto the surf aces of an organ, peritoneal, etc. Specific examples ofthis use are provided below in 1-1, 1-4, 1-5 and 1-6.

(2) The Coverage (Application Method) as a Tissue ReconstructionMaterial/Anti-Adhesion (Application Purpose)

Use (usage) of the composition for the purpose of suppressing theformation of adhesion with surrounding tissues by applying an amniononto the surfaces of an organ, peritoneal, etc. Specific examples ofthis use are provided below in 2-1, 3-1, 3-2, and 3-3.

(3) The Indwelling (Application Method) of a Tissue ReconstructionMaterial/Anti-Adhesion (Application Purpose)

Use (usage) of the composition for the purpose of suppressing theformation of adhesion by indwelling an amnion at the site where anadhesion is formed at a greater frequency. Specific examples of this useare provided below in 1-2, 1-3, and 2-2. Many of conventionalanti-adhesives take this form. For example, Seprafilm currently used asan anti-adhesive takes the similar form to 1-2 below. However, Seprafilmcannot be used in the applications 1-3 and 2-2below.

Exemplary applications of the sheet-shaped composition as tissuereconstruction material will now be specifically described (See FIG. 1).

1. Application in the Field of Digestive Surgery

1.1 Application in reconstruction of damaged organ and anti-adhesion

Various surgical operations in general leave a minute damage in organsduring the procedure. Unless appropriately restored at any early stageafter the surgery, destroyed chorionic structure of the damaged lesiontends to form an adhesion between organs, and may occasionally result ina loss of basic functions. Such a problem can be solved by making themost of amniotic abilities to reconstruct tissues and to preventadhesion. Specifically, the tissue reconstruction material is used tocover damaged surface of an organ to facilitate tissue reconstructionand prevent adhesion. For such a purpose, the tissue reconstructionmaterial constructed with, for example, cryopreserved amnion with anepitheium, cryopreserved amnion without an epithelium, lyophilizedamnion with an epithelium, amnion with adhesive components attached butwithout an epithelium, and so on can be favorably utilized. Although itis preferable to use tissue reconstruction materials constructed with adesiccated amnion (for example lyophilized amnion with an epithelium,lyophilized amnion without an epithelium) because of its handlingreadiness, those which are constructed with lyophilized amnion (forexample, lyophilized amnion with an epithelium, lyophilized amnionwithout an epithelium) can be selected for use in the area, such asheart, where the carrier requires a resiliency. As for the applicationmethod, the tissue reconstruction material is placed in position uponcompletion of a surgery such that it directly covers the damaged area oforgan with its amniotic basal membrane facing the peritoneal cavity,and, if necessary, is immobilized, for which a suture thread such asvicryl can be used. When a tissue reconstruction material constructed bya desiccated amnion is used, the immobilization process such as suturecan be done without since a high avidity is expected. Similarly, atissue reconstruction material constructed together with adhesivecomponents is also expected to have a high avidity. Thus, it ispreferable that immobilization of the tissue reconstruction materialonto any applied site can be attained without any specific suture orother immobilization process since such a suture or other immobilizationmakes the procedure laborious, and may induce inflammation therebypromoting the formation of adhesion. In particular, use of tissuereconstruction material constructed with lyophilized amnion with anepithelium is most preferable since it exerts a high avidity towardapplied site without any possibilities of eliciting a foreign bodyreaction against the adhesive components.

The procedure described above is expected to provide reconstruction ofchorionic structure at an early stage after surgery, and to attainreconstruction of damaged organ while preventing adhesion.

1-2. Damaged Organ—Application for preventing adhesion between wounds.

Various surgical operations occasionally leave adhesion betweenintraperitoneal organ and wound. Adhesion of the intraperitoneal organto wound physically fixes the organ, abrogates its mobility and causesclogging of the internal cavity, resulting in paralytic enterostasis.The capacity of amnion to prevent adhesion can be used to solve theproblem. For such a purpose, tissue reconstruction materials constructedwith cryopreserved amnion with an epithelium, cryopreserved amnionwithout an epithelium, lyophilized amnion with an epithelium, reinforcedhybrid amnion, etc can be utilized. Since strength sufficient to preventwrinkles is required, tissue reconstruction materials constructed withdesiccated amnion is preferably used, with tissue reconstructionmaterials constructed with desiccated and reinforced hybrid amnion beingmore preferred. The application methods are as follows. Upon completionof a surgical operation, a tissue reconstruction material is insertedimmediately beneath a wound, and left indwelt. The tissue reconstructionmaterial is placed in position such that the basal membrane side of itsamnion faces peritoneal side, and chorionic membrane faces abdominalwall. After the application, it may be fixed by, for example, suture,though it is preferred to have it simply left indwelt without anyfixation.

The above procedure can successfully attain prevention of adhesionbetween an organ and a wound after a surgery.

1-3. Application to Prevention of Adhesion to Pelvic Floor

Colon, uterine and other extraperitoneal organs a facial part of whichare not covered by peritoneal reside in intrapelvic space. Surgery onsuch an extraperitoneal organ may occasionally produce in the pelvicspace where no peritoneal exists, allowing small intestine to collapseon pelvic floor to form an adhesion with pelvic wall. Since theadhesion, once formed, is typically difficult to detach, anypreventative procedure is essential. The ability of amnion toreconstruct chorionic membrane can be used to prevent adhesion of pelvicfloor. For such a purpose, tissue reconstruction materials constructedwith, for example, cryopreserved amnion with an epithelium,cryopreserved amnion without an epithelium, lyophilized amnion with anepithelium, and reinforced hybrid amnion can be favorably used. Theproperties required for the tissue reconstruction materials are similarto those referred to in 1-2. The application method is as follows.Tissue reconstruction material is inserted into pelvic floor uponcompletion of a surgical operation, and lightly pressed onto peritonealto ensure coverage. The tissue reconstruction material is placed suchthat the basal membrane side of the amnion faces abdominal cavity andthe chorionic membrane side faces peritoneal. After the placement, thetissue reconstruction material can be immobilized by means of, forexample, suture, it is preferably left unimmobilized, only serving tocover.

The above procedure can successfully attain prevention of adhesion topelvic floor after a surgery.

1-4. Application to Reconstruction of Peritoneal Wall

Peritoneal wall may be damaged to have a defect by a plurality ofsurgeries or peritoneal diseases such as abdominal incisional hernia.Amnion can be used as a carrier for complementing damaged peritoneal.For such a purpose, tissue reconstruction materials constructed with,for example, cryopreserved amnion with an epithelium, cryopreservedamnion without an epithelium, lyophilized amnion with an epithelium, andamnion with adhesive components attached but without an epithelium canbe favorably used. If the defect of the peritoneal is extensive andsevere, it is preferable in respect of strength to use a tissuereconstruction material constructed with cryopreserved amnion with anepithelium. It is subject to application upon completion of the surgery.The tissue reconstruction material is first put on the defect area ofthe peritoneal so as to cover that area, with the basal membrane side ofthe amnion facing peritoneal cavity, and then, alternatively, the tissuereconstruction material may be immobilized by means of, for example,suture. If a tissue reconstruction material constructed with amnion withadhesive components attached thereto is used, those adhesive componentsmay attain immobilization. The above procedure is expected to attainreconstruction of abdominal wall.

1-5. Application to Suppression of Peritoneal Metastasis

Peritoneal metastasis is a case where metastasis is caused byprogression of stomach cancer, colon cancer and ovarian cancer resultingin cancer cells' spread from a particular tissue through pleural fluidor ascites fluid into coelom. Since cancers that accompany peritonealmetastasis have extremely poor prognosis, methods for suppressingmetastasis have been in demand. However, any effective measures thatmeet such a demand are still unknown. The properties of amnion areexpected to be exploitable to suppress metastasis. Dorsal mesogastrium,diaphragm, and mesentery, amongst others, are known as sites wherecancer cells cause metastasis at a greater frequency. These milky spots,so to speak, can be pie-covered with tissue reconstruction material toform a barrier to successively attain suppression of metastasis. Forsuch a purpose, tissue reconstruction materials constructed with, forexample, cryopreserved amnion with an epithelium, cryopreserved amnionwithout an epithelium, lyophilized amnion with an epithelium, and amnionwith adhesive components attached but without an epithelium can befavorably used. However, use of tissue reconstruction materialsconstructed with lyophilized amnion with an epithelium is preferredbecause of their readiness in handling. As an exemplary applicationmethod, the tissue reconstruction material can be placed such that itwraps around the tissue of interest and covers the application site.Alternatively, the tissue reconstruction material can be immobilized onthe application site by means of suture with a patch intervening aportion of it, or by means of adhesive components attached onto theamnion. The timing for application of the tissue reconstruction materialmay be after metastasis as a result of progression of cancer, or beforeany spread (Preliminary use).

1-6. Application to Prevention of Recurrent Adhesion

Ileus may be caused after laparotomy, by adhesion between bowels orbetween a bowel and abdominal wall, leading to a bending thereaboutwhich causes passage disorder and resulting dysfunction. Even if theadhesion is successfully disrupted, the once adhered tissues, especiallythose tissues which caused a severe inflammation and cicatrized havedefective chorionic membrane, and, therefore, typically causere-adhesion post surgery. Amnion can be used to prevent re-adhesion postsurgery, and facilitate repair and reinforcement of the defectivechorionic membrane. For such a purpose, tissue reconstruction materialsconstructed with, for example, cryopreserved amnion with an epithelium,cryopreserved amnion without an epithelium, lyophilized amnion with anepithelium, and amnion with adhesive components attached but without anepithelium can be favorably used. However, use of tissue reconstructionmaterials constructed with lyophilized amnion with an epithelium ispreferred because of their readiness in handling. As an exemplaryapplication method, after laparotomy and when the adhesion has beenphysically disrupted, the tissue reconstruction material can be placedsuch that it wraps around the bowel like a tube. The tissuereconstruction material is placed in position such that the basalmembrane side of the amnion faces abdominal cavity. Immobilization thattypically occurs after that placement may be in the form of suturebetween the organ and the amnion or between the amnions (making theamnion a tube) using a suture such as vicryl. Alternatively, the tissuereconstruction material may be left indwelt without any suture. Whentissue reconstruction materials constructed with an amnion with adhesivecomponents attached thereto is used, those adhesive components may serveto attain the immobilization. It is preferable that immobilization ofthe tissue reconstruction material to the applied site is attainedwithout a separate immobilization process such as suture, since such asuture or other immobilization makes the procedure laborious, and mayinduce inflammation thereby promoting the formation of adhesion. Inparticular, use of tissue reconstruction material constructed withdesiccated amnion is most preferable since it exerts a high aviditytoward applied site without any possibilities of eliciting a foreignbody reaction against the adhesive components.

The procedure described above is expected to provide reconstruction ofchorionic structure at an early stage after surgery while preventingre-adhesion.

2. Application in Obstetrics and Gynecology 2-1. Application toSalpingemphraxis

Adhesion of salpinx to organs such as peritoneal causes blockage of thesalpinx and makes oval passage difficult, resulting in infertility.Amnions can be used to prevent adhesion of salpinx. Tissuereconstruction Materials of the invention can be applied for such apurpose in the following procedure. Adhesion is firstly disrupted and atypical fimbrioplasty is performed. After the surgery, but beforeclosing the operative wound, the area of salpinx is covered with tissuereconstruction material. For such a purpose, tissue reconstructionmaterials constructed with, for example, cryopreserved amnion with anepithelium, cryopreserved amnion without an epithelium, lyophilizedamnion with an epithelium, and amnion with adhesive components attachedbut without an epithelium can be favorably used. However, use of tissuereconstruction materials constructed with lyophilized amnion with anepithelium is preferred because of their readiness in handling. As anexemplary application method, the tissue reconstruction material can beplaced such that it wraps around the tissue of interest and covers theapplication site. Alternatively, the tissue reconstruction material canbe immobilized on the application site, by means of suture with a patchintervening a portion of it, or by means of adhesive components attachedonto the amnion.

2-2. Application to Prevent Adhesion of Pelvic Floor

Uterine inside pelvis is an extraperitoneal organ, and an approximately50% of its surface is not covered with peritoneal. Therefore,hysterectomy produces sites where no peritoneal exists, causing adhesionbetween pelvic floor and small intestine. Amnions can be used to preventthe adhesion of pelvic floor. The form and application methods of thetissue reconstruction materials used for such a purpose are similar tothose described in 1-3.

3. Application to Ophthalmic Field 3-1. Application to Surgery to TreatGlaucoma

Glaucoma is a disease where optic nerves are affected to cause narrowingof visual field and low vision. Glaucoma is treated by severingtravecula to form a new auieous humour discharge system. However, suchan operation occasionally results in adhesion between sclera andconjunctiva and produces a poor outcome. Use of amnions may be effectivefor such a problem. In particular, after a typical operation of severingtravecula, the tissue reconstruction material is inserted beneath theconjunctiva. for such a purpose, tissue reconstruction materialsconstructed with, for example, cryopreserved amnion with an epithelium,cryopreserved amnion without an epithelium, lyophilized amnion with anepithelium, and amnion with adhesive components attached but without anepithelium can be favorably used. However, use of tissue reconstructionmaterials constructed with lyophilized amnion with an epithelium ispreferred because of their readiness in handling. After the application;the tissue reconstruction material may be immobilized to applied siteby, for example, suture.

3-2. Application to Symblepharon

Symblepharon is a disorder where cicatrices are formed between palpebralconjunctive and eye ball, causing adhesion between eyelid and eye ball.Symblephalon typically accompanies an extensive damage on eye surface,and any disruption of adhered tissue entail recurrence of thesymblephalon. Amnions can be used to suppress symblepharon. As anexemplary application method, the adhesion lying between eyelid and eyeball is disrupted, and the cicatrized conjunctival tissue is detached,thereby exposing sclera, which is then covered with tissuereconstruction material. For such a purpose, tissue reconstructionmaterials constructed with, for example, cryopreserved amnion with anepithelium, cryopreserved amnion without an epithelium, lyophilizedamnion with an epithelium, and amnion with adhesive components attachedbut without an epithelium can be favorably used. However, use of tissuereconstruction materials constructed with amnion with adhesivecomponents attached without an epithelium is preferred, for example,because of their readiness in handling. Immobilization of the usedtissue reconstruction material onto applied site is attained typicallyby the adhesive components. The site, covered by the tissuereconstruction material, may be on either side of eyelid or sclera.

3-3. Application to Recurrent Pterygium

Pterygium is a disorder where conjunctival tissue cause an abnormalhyperplasia, and those hyperplastic tissues adhere to corneal, resultingin astigmatism and low vision. Amnions may be effective for thisdisorder. As an exemplary application method, pterygium tissue isdisrupted to expose sclera, which is then covered with tissuereconstruction material. For such a purpose, tissue reconstructionmaterials constructed with, for example, cryopreserved amnion with anepithelium, cryopreserved amnion without an epithelium, lyophilizedamnion with an epithelium, and amnion with adhesive components attachedbut without an epithelium can be favorably used. However, use of tissuereconstruction materials, which are constructed with adhesive componentsattached without an epithelium, is preferred because of their readinessin handling. Immobilization of the used tissue reconstruction materialonto applied site is attained typically by the adhesive components.

B. Application of the sheet-shaped composition comprising cell layer

Those sheet-shaped compositions, which comprises cell layer, can beapplied to reconstruction of corneal and retina (in treatment of, forexample, Stevens—Johnson syndrome, thermo-chemical injuries, ophthalpemphigoid, ablatio retinae, degeneratio maculae luteae senil, glaucoma,and degeneratio pigmentosa retinae), treatment of epidermal diabeticulcer (epidermis), bullous epidermolysis, or ambustion.

(Producing Method of Sheet-shaped Composition)

Another embodiment of the present invention relates to a producingmethod of sheet-shaped composition. The producing method according tothe invention comprises the following steps of; (a) preparing an amnion,and (b) adding trehalose to the amnion.

1. Preparation of Amnion: step (a)

“Amnion” is a membrane covering the outermost layer of the uterus andthe placenta in mammals, and including a basal membrane and anepithelium layer formed on parenchymal tissue rich in collagen. It ispreferable that human amnion is used as amnion. Human amnion can becollected by, for example, human embryonic membrane, placenta, etc.obtained at the time of afterbirth at delivery. Specifically, the humanamnion can be prepared by treating and purifying the integrated materialincluding human embryonic membrane, placenta, and umbilical cordobtained right after delivery. The treating and purifying method canemploy a well-known method, for example, a method described in JapanesePatent Unexamined Publication No. H5-5698, etc. That is to say, amnionis detached from the embryonic membrane obtained at delivery andremaining tissue is removed by a physical treatment such as ultrasoniccleansing and an enzyme treatment, and the like. Then, appropriatewashing process is carried out and thus the human amnion can beprepared.

The thus prepared human amnion can be cryopreserved before use. Thehuman amnion can be frozen in a liquid mixing equal volume ratio of DMEM(Dulbecco's modified Eagle's medium) and glycerol at, for example, −80°C. By cryopreservation, not only the improvement in operation propertybut also reduction of the antigenicity can be expected.

Intact amnion may be used but it is preferable that amnion from whichepithelium has been removed by a scraping treatment, etc. is used. Byremoving the epithelium, antigenicity is reduced. For example,cryopreserved human amnion is thawed and then subjected to a treatmentwith EDTA or proteolytic enzyme so as to loosen the adhesion betweencells. Then, epithelium is scraped by using a cell scraper, etc. Thus,the human amnion from which epithelium has been removed can be prepared.

Preferably, an epithelium of amnion is removed by a method comprisingthe following step of;

(1) preparing an amnion isolated from an organism;(2) subjecting the amnion to freeze and thawing process;(3) subjecting the amnion post the freeze and thawing process to tryptictreatment; and(4) washing the amnion post the tryptic treatment.

Similarly to the conventional, manual removal of epithelium, the removalof epithelium according to the present invention allows a completeremoval of epithelium with minimal damage on basal membrane. Inparticular, the removal of epithelium allows a complete removal ofepithelium, while providing an amnion having a basal membrane with itsnative'structure favorably maintained. Such an amnion can servefavorably, for example as medium (base) for culturing cells. On theother hand, the following method of removing an epithelium is extremelyready to handle and less time-consuming, relative to conventional,manual removal. Moreover, it facilitates to treat a multiple of amnionat a time. Further, since it requires no special skills, automationthereof is ready.

Each step of the instant method for removing epithelium will bedescribed below in detail.

(Preparation of Amnion: Step 1)

In step (1), an amnion is prepared, which is herein preferably humanamnion. Human amnion can be harvested from, for example, human fetalmembrane or placenta obtained during afterbirth of a delivery.Specifically, a solid mass comprising human fetal membrane, placenta andumbilical cord obtained immediately after a delivery is treated andpurified to prepare human amnion. Such a method for preparing humanamnion may be performed by any known method, such as that described inJapanese Patent Publication No. 5-56987. These steps are performedtypically in the following procedure.

(1) Harvesting of Amnion

A part of placental tissue is harvested during delivery, and an amniotictissue is manually detached from that placental tissue. Alternatively,the amniotic tissue may be temporarily frozen.

(2) Removal of Blood Cell Components and Others

Any blood cell components that are left on the amnion is washed awaywith physiologic saline and removed. In addition, chorionic membrane ismanually detached and removed. Thus, although it is preferable to makethe amnion free from blood cell components and chorionic membrane atthis stage, the removal of blood cell components and/or detachment ofchorionic membrane may occur after a freeze-thawing process (step 2).

The human amnion thus prepared can be frozen and stored until nextprocess. The freezing of human amnion can be performed at −80° C. in amixture of DMEM (Dulbecco's modified Eagle's medium) and glycerol in anequivalent amount in volumetric ratio. Cryopreservation is expected tonot only improve handling readiness, but also reduce antigenicity.

(Securing into Frame)

The amnion prepared in the above procedure is preferably secured into aframe, and subjected to following process since by securing into aframe, the amnion is made ready to handle.

Exemplary methods of securing the amnion are shown in FIG. 2. In FIG. 2a, two pieces of frame (1, 2) are used. An amnion 10 is spread wide andsecured into the two frames with its edge clamped between the frames. InFIG. 2 b, a frame 3 and a plate-like member 4 are used to secure anamnion 10. The amnion 10 is placed and spread wide on the plate-likemember 4, with upper side of the amnion 10 facing upward. Then; theframe 3 is mount over the amnion 10, clamping the edge of the amnion 10between the plate-like member 4 and the frame 3. As a result, only theepithelium side of the amnion 10 is exposed. Accordingly, only theepithelium side of the amnion 10 can be brought into contact withtryptic solution in the following tryptic treatment (for example byadding tryptic solution inside the frame 3). This enables the tryptictreatment to be performed without affecting other portions (stratumcompactum and basal membrane of the amnion) than the epithelium. Inother words, the epithelium of the amnion can be subjected to thetryptic action, while protecting stratum compactum and other portion ofthe amnion against the tryptic action.

(Freeze-Thawing Process: Step 2)

In this step, the amnion is temporarily frozen, and then thawed. Thisfreeze-thawing process facilitates removal of the amniotic epithelium inthe following tryptic treatment. This is thought to be due to looseningof the adhesion (binding state) between the amniotic epithelium and thebasal membrane.

Freezing temperature may be in the range of about −20° C. through about−80° C. In consideration of sufficient freezing condition andavailability of universal freezer, freezing at about −80° C. ispreferable. On the other hand, thawing may be at a temperature in therange of about 4° C. through about 50° C. The thawing temperature ispreferably about 37° C.

It is preferable to repeat the freeze-thawing process. Repetition ofthis process adds to the effect of freeze-thawing process byfacilitating the removal of epithelium in the following tryptictreatment. However, it is expected that the repetition beyond what isnecessary will adversely affect any portions other than epithelium.Accordingly, the freeze-thawing process is preferably repeated in therange of two through four times. The inventions have found thatrepetition of freeze-thawing process twice with freezing at −80° C. andthawing at 37° C. yields a necessary and sufficient condition. Based onthis knowledge, it is believed that, under the condition of freezing at−80° C. and thawing at 37° C., repetition of freeze-thawing twice ispreferable.

The conditions for each round of repeated freeze-thawing. process(freezing temperature and thawing temperature) may be wholly the same,partly the same, or totally different. In view of handling readiness,the conditions are preferably the same.

(Tryptic Treatment: Step 3)

In this step, the amnion post the freeze-thawing process is treated withtrypsin. The trypsin treatment is performed by contacting the amnionwith tryptic solution. An exemplary tryptic solution is that with atryptic concentration of about 0.01% (w/v) through about 0.05% (w/v).Preferably, a tryptic solution at a tryptic concentration of about 0.02%(w/v) is used. If the tryptic concentration of a tryptic solution is toolow, the tryptic action is not sufficiently exerted. On the other hand,if the tryptic concentration is too high, the tryptic action is exertedfavorably on the amniotic epithelium, but inadvantageously extendsbeyond the epithelium to damage the underlying amniotic stratumcompactum and basal membrane.

Trypsin may be of any origin, including bovine, porcine, human and anyother origins commercially available. Trypsin-EDTA (Invitrogen), andTrypsin 1:250 (Sigma), for example, can be favorably used.

Tryptic solution may typically have any chelators added thereto, whichis not always necessary. Exemplary chelators are EDTA, NTA, DTPA, EDTA,GLDA or any combination thereof. Chelators may be at concentration of,for example, about 0.1 mM through about 0.6 mM.

It is preferable that the tryptic treatment is performed under suchconditions that only the epithelial side of the amnion is brought intocontact with tryptic solution in order to protect other portions thanthe amniotic epithelium against tryptic action. The epithelial side ofthe amnion exclusively can be brought into contact with tryptic solutionby, for example, immersing the amniotic epithelial side in trypticsolution, adding or applying tryptic solution onto the amnioticepithelial side, or blocking the chorionic side of the amnion, so as notto contact with tryptic solution before immersing wholy in trypticsolution. As described above, use of the amnion pre-secured into a frameas shown in FIG. 2 b (framed amnion) can attain the exclusive contact ofthe epithelial side of the amnion with tryptic solution, for example byimmersing the framed amnion in tryptic solution since only theepithelial side is exposed. This method also has an advantage ofsimplifying the tryptic treatment by making it a simple operation ofimmersing a framed amnion. In addition, use of a framed amnion can takeother various forms of tryptic treatment than immersing the framedamnion, wholly along with the frame, into tryptic solution, for exampleby immersing only the epithelial side of the amnion in tryptic solution(for example by facing the epithelial side of the amnion downward to beimmersed in tryptic solution), by adding tryptic solution into theframe, or by applying the'tryptic solution onto the epithelial side ofthe amnion, to bring only the epithelial side into contact with trypticsolution.

The time period for tryptic treatment (time period for contacting withtryptic solution) may be, for example, in the range of about 5 minutesthrough about 60 minutes. Preferably, the time period is from about 10minutes to about 20 minutes, more preferably about 15 minutes. If thetreatment time period is too short, tryptic action is not sufficientlyexerted, resulting in an insufficient removal of the amnioticepithelium. On the other hand, if the time period is too long, thetryptic action may extend to and damage basal membrane and stratumcompactum of the amnion.

The temperature at which the tryptic treatment is performed by fromabout 25° C. to about 42° C. such that trypsin acts favorably.

During the contact of tryptic solution, it is preferred to maintain theamnion at a stand-still condition under which tryptic solution mayhardly permeate through basal membrane and stratum compactum.Alternatively, the tryptic treatment may be performed in a plurality ofsteps.

(Washing: step 4)

After the amnion is contacted with tryptic solution in the mannerdescribed above, it is subjected to washing wherein the tryptic solutionattached is removed and, at the same time, so is the amniotic epithelium(epithelial cells). This washing of the amnion after tryptic treatmentmay be done by leaving it under an appropriate stream of solution (forexample running water), by shaking (for example shaking up and down) itwhile immersing it in an appropriate solution and, or by subjecting itto ultrasound or other vibration while immersing it in an appropriatesolution. The washing solution may be, for example, saline solution,phosphate buffered saline, pure water and DMEM.

The amnion after washing may be stored in a refrigerator or freezeruntil use. For example, the amnion can be stored with it immersed in asolution containing glycerol (for example, DMEM (Dulbecco's ModifiedEagle Medium: GIBCOBRL) containing 50% glycerol).

2. Addition of Trehalose: step (b)

Addition of trehalose to the amnion can be performed by immersing theamnion in trehalose solution. For example, the amnion is immersed in asolution of 5% (w/v)-20% (w/v) trehalose in distilled water or phosphatebuffered saline (PBS(-)). The temperature during the immersion is, forexample, from about 4° C. to about 37° C. The immersion time period is,for example, from about one hour to one day. The trehalose used may be,for example, “Toreha” (Registered Trademark) available from HayashibaraCorp. or “Torehainochi” available from H plus V Lifescience Corp.

Addition of trehalose to the amnion can take other forms such as, forexample applying trehalose solution to the amniotic surface, sprayingtrehalose solution to the amniotic surface and adding trehalose directlyonto the amniotic surface.

The step of removing an epithelium from the amnion (for example thesteps 2 through 4 above) may .have a preceding step of adding trehalose.

3. Freezing Process or Desiccation Process: Step (c)

In one embodiment of the invention, the amnion, once added withtrehalose, is frozen or desiccated. This process improves thepreservability and handling readiness. Moreover, the desiccation processgreatly enhances the preservability and handling readiness of resultingsheet-shaped composition. Further, a change of the surface profile ofthe amnion that accompanies the desiccation is expected to crease theaffinity (adhesiveness) of the amnion to tissues of a living organism.The desiccation process of the amnion is preferably performed throughlyophilization since this process mitigates the reduction of amnioticflexibility. In addition, lyophilization process is preferable inrespect of maintaining the structure of the amniotic basal membranecomponents. The lyophilization process removes the moisture contained ina frozen sample (for example, a sample frozen at about −40° C.) throughsublimation under a low atmospheric condition (vacuum) where a boilingpoint lies in the range of about −20° C. (107 Pa, 0.8 Torr) throughabout −50° C. (4 Pa, 0.03 Torr). Since lyophilization dehydratesuniformly from inside, and achieves a high dryness, native function andprofile can be highly maintained even after the desiccation. Further,lyophilization has advantages of, for example, 1. having a lessdeterioration during the process, 2. being ready to making it aseptic,4. and yielding an improved desiccated result which has a high abilityto regain its original profile.

Lyophilization can be performed by a lyophilizer comprising a vacuumchamber, cooling and heating apparatus, and exhauster (cold trap andvacuum pump). A numerous lyophilizers are commercially available, any ofwhich can be utilized for performing the instant lyophilization. Theconditions for the lyophilization can be set according to anyinstruction attached to the lyophilizer used and in .consideration ofthe size and a desired dryness of the sample that undergoes thedesiccation. The dryness may be set such that, for example, its wateractivity (AW) becomes less than 0.5.

Desiccated amnion with a desired size and shape can be obtained bysevering or cutting out the desiccated amnion. The desiccated amnionthus obtained may be secured to a support or frame.

In one embodiment of the present invention, the desiccated amnionobtained through the desiccation process is contained in any suitablecontainer such that there is substantially no contact with oxygen.Containment into a package in such a state substantially sequesteredfrom oxygen attains a non-epithelium-containing desiccated amnion havingan extremely high preservability.

For example, the amnion after desiccation is contained in a suitablecontainer and, for example, the air inside the container aspired andremoved to evacuate or replace with nitrogen, thereby putting the amnioninto a packaged substantially sequestered from oxygen. Alternatively,the container may also contain deoxidant to remove the remaining oxygen.These methods may optionally be combined. An exemplary container is, forexample, a bag- or tube-like container (two sheets may be superposed toeach other with their periphery sealed) made of plastic synthetic resinor a bottle-like container made of glass or other inorganic material.

The freezing process or desiccating process may be preceded or followedby a step of covering the chorionic side of the amnion withbioabsorbable material to strengthen the amnion. Exemplary bioabsorbablematerials are polyglactin 910, gelatin, collagen, and poly-lactic acid.

4. Sterilization Process Step: Step (d)

Sterilization process minimizes the risk of bacterial contamination. Forexample, EOG (Ethylene oxide gas), UV (Ultraviolet), γ-ray treatment canbe used to sterilize the amnion. γ-ray sterilization is most preferableamongst these because of its low tendency to decrease amniotic physicalproperties. Dose for the γ-ray sterilization may be, for example, from 2kGy to 50 kGy, preferably from 10 kGy to 30 kGy, more preferably from 15kGy to 5 kGy. It is preferable to perform the sterilization processafter the amnion which underwent the series of process has beencontained in a container or wrapped in a film or sheet, etc.Accordingly, the sterilization process is preferably preceded by a stepof containing the amnion in a container, etc. The amnion is preferablycontained or wrapped under a condition having substantially no contactwith oxygen for minimizing deterioration of quality and allowing storagefor a longer period of time.

5. Attachment of Adhesive Components: Step (e)

In one embodiment of the invention, fibrinogen and thrombin are attachedto the surface of the amnion. The attachment of these components can beperformed after the desiccation process described above. Use ofdesiccated amnion allows for a favorable attachment of fibrinogen andother adhesive components.

The attachment of fibrinogen and thrombin to the surface of the amnionis performed independently or simultaneously. Methods of attachment arenot limited. An exemplary method of attachment is by applying, droppingor spraying a solution of the attached components to the amnioticsurface, or by immersing the amnion in a solution of the attachedcomponents. Alternatively, fibrinogen itself (or thrombin itself) or anycomponents deposited after dissolving fibrinogen (or thrombin) in anappropriate solvent is added (sprinkled) on the amniotic surface toattach fibrinogen (or thrombin) to the amniotic surface.

Preferably, a mixture of these two components is prepared and, forexample, by applying or dropping the mixture to attach fibrinogen andthrombin to the amniotic surface. Specific exemplary methods ofsimultaneously attaching the two components are described below.

A solution of fibrinogen is first prepared. Specifically, fibrinogen isdissolved in ethanol (for example 94% ethanol) or other solvent (solventmedium) at a desired concentration. Besides ethanol, alcohols such asanhydrous ethanol, isopropanol, methanol and acetone can be used assolvent. Meanwhile, thrombin solution is prepared separately in the samemanner. Exemplary solvents that can be used in this case are ethanol(such as 99.5% ethanol), anhydrous ethanol, isopropanol, methanol andother alcohols and acetone.

Next, the fibrinogen solution and thrombin solution prepared in theabove described manner are mixed. The mixture thus obtained is used toperform applying, dropping or other procedure on the amnion as describedabove. If the described mixture of fibrinogen solution and thrombinsolution is used for the attachment procedure, it is preferable toensure that the water content in the mixture is at too high a level. Ifthe water content is too high, a reaction between fibrinogen andthrombin occurs before the attachment procedure, hampering thatprocedure. In addition, in order to attain a favorable adhesivenessafter transplant, the fibrinogen and thrombin attaching to the amnionhas preferably no preceding action therebetween. In consideration of theabove factors, a solvent for each of fibrinogen and thrombin ispreferably water-soluble and volatile, and has less water content.

While application or dropping, for example, of fibrinogen solution andthrombin solution, or a mixture of fibrinogen and thrombin is typicallyperformed uniformly over the entire region of the amniotic surface, itmay be performed on a limited region (for example by spotting on aplurality of regions with a distance therebetween, or by placing themonly over the periphery), or with their attached density varied.

In the above procedure, the attachments of fibrinogen and thrombin areperformed simultaneously. However, each of these components may beattached in separate steps. Specifically, attachment of fibrinogen andattachment of thrombin may take place in two steps. However, in respectof simplifying the procedure and attaining a uniform distribution ofattached fibrinogen and thrombin, it is preferable to use a mixture offibrinogen and thrombin to perform the attachment in one single step.

Fibrinogen and thrombin can be prepared from blood according toconventional methods. Alternatively, recombinant fibrinogen and othercomponents can be used, in which case any appropriate culture solutionor lysis solution of cultured cells can be used according toconventional methods. Alternatively, any commercially availablefibrinogen, or other components can be used. For example, fibrinogenderived from human can be purchased from Baxter Corp. Similarly,thrombin derived from human can be purchased from Baxter Corp.

In addition to fibrinogen and thrombin, aprotinin may be attached to theamniotic surface. Specifically, in the instant embodiment, a step ofperforming attaching aprotinin (step b-1) is further performed. Theattachment of aprotinin can be performed in the similar means andprocedure to the attachment of fibrinogen, etc. Specifically,application, dropping, spraying, immersion and other procedure using theaprotinin solution results in attachment of aprotinin to the amnioticsurface. The aprotinin solution can be prepared by dissolving aprotininin sodium chloride solution (for example 0.85% solution), potassiumchloride solution, calcium chloride solution, magnesium chloridesolution, etc.

Aprotinin can be prepared from bovine pancrea according to conventionalmethods. Alternatively, recombinant aprotinin can be used, in which caseany appropriate culture solution or lysis solution of cultured cells canbe used according to conventional methods. Alternatively, anycommercially available aprotinin may be used. For example, aprotinin ofbovine origin can be purchased from Bayer Pharmaceuticals. Although thestep of attaching aprotinin can be performed singularly, the steps ofattaching fibrinogen and thrombin are preferably performedsimultaneously since the procedure for attaching adhesive components isfacilitated as a whole. It is further because that a more uniformdistribution of fibrinogen, thrombin and aprotinin attached on theamniotic surface can be attained. For example, by preparing a mixture offibrinogen, thrombin and aprotinin and, for example, applying themixture, simultaneous attachment of these components to the amnion canbe attained. The order of mixture of these three components is notlimited.

The attachment of fibrinogen, etc. is performed on either or both sidesof the amnion. In the former case, the attachment of fibrinogen, etc. isperformed on the surface (i.e. chorionic side) opposite to theepithelium (the side where an epithelium was present), irrespective ofthe presence or absence of the epithelium on the amnion.

After fibrinogen and thrombin (plus aprotinin in some occasion) havebeen attached, desiccation process is performed, as necessary, to yielda sheet-shaped composition with a high stability in storage and with aform favorable for handling (transportation, transplant, etc.).

The desiccation process may adopt any typical desiccation procedure,such as air-drying, vacuum-drying, suction-drying, lyophilization, andso on.

(Method for Producing Sheet-Shaped Composition Comprising Cell Layer)

In one embodiment of the invention, cell layer utilizing tissue-derivedcells are formed on the amnion. The step of forming cell layer can beperformed in the following procedure. Any appropriate tissue-derivedcells are prepared (step of preparing tissue-derived cells). Thetissue-derived cells are selected so as to accommodate the applicationof resulting sheet-shaped composition. For example, in order to producea sheet for reconstructing skin epidermal tissues, skin epidermal cells(including their stem cells and precursor cells) and follicularepithelial cells are preferably used. Similarly, in order to reconstructcorneal epithelial tissues, corneal epithelial cells (including theirstem cells and precursor cells) are preferably used, and, in order toreconstruct mucosal epidermal tissues, mucosal epithelial cells(including their stem cells and precursor cells) are preferably used.Exemplary mucosal epithelial cells are oral mucosal epithelial cells,intestinal mucosal epithelial cells and air duct mucosal epithelialcells.

Methods for preparing tissue-derived cells are hereinafter described,taking examples of skin epidermal cells, corneal epithelial cells, oralmucosal epithelial cells, intestinal mucosal epithelial cells, and airduct mucosal epithelial cells.

(Skin Epidermal Cell)

Firstly, when the skin is collected, a site to be collected isdisinfected with disinfectant such as povidone iodine prophylacticallyin advance and antifungal agent is externally applied thereto, followedby collecting a small skin piece in accordance with skin biopsy. Inculturing epidermal keratinocytes, fatty tissue and dermis are removedfrom the skin piece as much as possible by using scissors and washedwith Dulbecco's phosphate buffer (PBS) several times and soaked in 70%ethanol for one minute for sterilization. The piece is cut into a stripshape, soaked in Dispase solution and stood still over night at 4° C.Then, epidermis is peeled off from the dermis. The peeled epidermis iswashed, followed by disentangling the epidermal piece so as to preparesuspending solution of epidermal keratinocyte. The cells are suspendedin a serum free culture medium and seeded on a collagen-coated dish, andsubculture is carried out.

(Corneal Epithelial Cell)

Corneal epithelial cells can be obtained from a corneal limbus tissue.For example, endothelial cells are peeled off and removed from corneallimbus tissue, and conjunctiva is excised so as to form a single cellsuspension. Then, this is preserved in a nitrogen tank, and then rapidlymelted at 37° C. so as to adjust a corneal epithelial cell suspendingsolution. If necessary, subculture is carried out. For subculture, forexample, EpiLife™ (Cascade), an MCDB153 medium (NISSUI PHARMACEUTICALCO., LTD.), which are serum free media, and media produced by modifyingthe amino acid composition, etc. of the above-mentioned media can beused.

(Oral Mucosal Epithelial Cell)

As the oral mucosal epithelial cells, cells existing in the dental rootpart (oral crevicular mucosal epithelial cells), cells of labial part,cells of palate part, cells of buccal part, and the like, can be used.Among them, it is particularly preferable to use oral crevicular mucosalepithelial cells because of the high proliferation ability and lowantigenicity. The oral mucosal epithelial cells can be collected byablating a site where targeted cells exist with the use of a scalpel, orby scraping it out. Oral crevicular mucosal epithelial cells can becollected by separating oral mucosal epithelial cells from the enamelcement transition portion and collecting the cells from the obtainedtissue piece. Note here that in order to remove impurities such asconnective tissue, preferably, a treatment with enzyme such as dispaseor trypsin, etc., filtration treatment are carried out.

(Intestinal Tract Mucosa Epithelial Cell)

The intestinal tract mucosa epithelial cells are collected fromintestinal tract epithelium tissue to an endoscope of the largeintestine, or by usual technique at the time of abdominal section.Furthermore, epithelial cells can be removed from tissue by lasercapture microdissection. The technique of the present invention can beapplied to sheet-shaped composition produced by using epithelial cellsfrom all the human digestive tract such as esophagus, upper stomach,duodenum, small intestine, and large intestine. When ulcer,inflammation, or the like, causes injuries of human digestive tractepithelium, cells derived from bone marrow play a roll as a rescue withrespect to emergency, so that the epithelium is repaired. The digestivetract epithelial cells, although a part of them, are also made from bonemarrow. In this sense, the present invention can be regarded to havesignificance that is equivalent to that using corneal epithelial cells.In general, an epithelial cell made of bone marrow, which is usuallyonly several cells per 1000 cells, are increased 50 to 100 times in theprocess in which ulcers (wounds) on the internal surface of thedigestive tract, which are generated by, for example, gastric ulcer andcolitis, are being cured. It is determined that about 1 of 10 digestivetract epithelial cells are derived from the bone marrow. Thesheet-shaped composition derived from the digestive tract mucosaepithelial cells are extremely significant because they urge theregeneration of intestinal tract epithelium with respect to ulcer andinflammation of intestine diseases which are designated intractablediseases, that is, severe intestinal tract infectious diseases such asulcerous colitis, Crohn's disease, Behchet's disease, and the like. Theeffectiveness with respect to intestinal tract allergy can be expected.

(Respiratory Tract Mucosa Epithelial Cell)

Respiratory tract mucosa epithelial cells can be easily obtained frombiopsy tissue of the respiratory tract mucosa. Similar to theabove-mentioned tissue, in order to remove impurities such as connectivetissue, it is preferable that treatment with enzyme such as Dispase,trypsin, and the like, or filter treatment is carried out. Therespiratory tract mucosa epithelial cells play an important role forpathologic conditions of various infectious diseases via biosynthesesand release of β defensin. Furthermore, respiratory tract mucosalepithelium also plays an important role in asthma or allergic disease.Providing sheet-shaped composition produced by the respiratory tractmucosa epithelial cells according to the present invention to therespiratory tract mucosa having tissue disorder would lead to not onlycarrying out emergency treatment but also providing artificialrespiratory tract. In particular, immunosuppression effect of the sheetwith amnion is useful.

It is preferable that after tissue is collected, oral mucosal epithelialcells, intestinal tract mucosa epithelial cells, and the like, aresubjected to a treatment with enzyme such as Dispase, trypsin, and thelike, or filter treatment in order to remove impurities such asconnective tissue.

It is preferable that the cells of biological origin are prepared from aperson (recipient) who undergoes transplantation. That is to say, it ispreferable that a donor of cells of biological origin is identical to arecipient of the sheet-shaped composition. By using such autologouscells, problem as to immunological rejection is avoided.

The prepared cells of biological origin are seeded onto amnion (step ofseeding cells of biological origin onto amnion), followed by culturingthereof (step of culturing and proliferating the seeded cells ofbiological origin).

In this embodiment, it is particularly preferable to use amnion fromwhich the epithelium has been removed. By removing the epithelium, thereduction of antigenicity can be expected. Furthermore, sinceunnecessary cells are removed in advance, target cell layers can beformed excellently. When amnion from which the epithelium has beenremoved is used, it is preferable that cells of biological origin areseeded on the side of the exposed surface from which the epithelium hasbeen removed (that is to say, side of the basal membrane). It is thoughtthat this side of the surface is rich in type IV collagen, so that theproliferation and stratification of the seeded cells of biologicalorigin can proceed excellently.

Herein, by using two types of cells, a hybridized cell layer may beformed. A method of forming a cell layer in such a case is described indetail hereinafter, taken the case where a sheet-shaped composition forreconstructing the corneal epithelium as an example.

Firstly, one of the cell types used for forming a cell layer (the firstcells), cells derived from mucosal epithelium such as oral mucosalepithelium, conjunctival epithelium, and nasal mucosal epithelium, orundifferentiated cells capable of constructing such mucosal epitheliumcan be preferably used. On the other hand, as the cell type (the secondcells) used for forming a cell layer together with the first cells,corneal epithelial cells, conjunctival epithelial cells, or amnionepithelial cells can be preferably used. These cells can be collectedfrom a living tissue in which these cells are present. Specifically, forexample, a part of the tissue in which target cells exist is collectedby using a surgical knife and the like, to the treatment such asremoving of the connective tissue, separation of cells, and the like,and formed in a state of the cell suspending solution (suspension). Notehere that as the first cells, two or more different types of cells maybe used. Similarly, as the second cells, two or more different types ofcells may be used.

It is suggested that oral mucosal epithelium that is suitable for acollection source of the first cells includes stem cells. Therefore, itis thought that the oral mucosal epithelium can easily carry outdifferentiation induction for forming cells capable of forming anepithelium-like cell layer. Furthermore, the use of oral mucosalepithelial cells has advantages that they are collected easily, a largenumber cells can be collected, and furthermore, even in the case oftreating corneal disease occurring in bilateral eyes, autologous cellscan be used so as to prepare a transplantation material, and the like.In particular, also fin patients from whom corneal epithelial cellscannot be collected, transplantation material derived from theautologous cells can be provided. This advantage is expected toradically dissolve the problem of clinically important rejection.

As the oral mucosal epithelial cells, cells existing in the dental rootpart (oral crevicular mucosal epithelial cells), cells of labial part,cells of palate part, cells of buccal part, and the like, can be used.Among them, it is particularly preferable to use oral crevicular mucosalepithelial cells because of the high proliferation ability and lowantigenicity. The oral mucosal epithelial cells can be collected byablating a site where target cells exist with the use of a scalpel, orby scraping it out. Oral crevicular mucosal epithelial cells can becollected by separating the oral mucosal epithelium that is attached toan extracted tooth from the enamel cement transition portion andcollecting the cells from the oral mucosal epithelium. Note here that inorder to remove impurities such as connective tissue, preferably, atreatment with enzyme such as dispase or trypsin, etc., filtrationtreatment are carried out.

Oral mucosal epithelial cells collected from a person other than thepatient who is intended to undergo a transplantation of the sheet-shapedcomposition of the present invention can be used. However, takenimmunorejection into consideration, it is preferable that oral mucosalepithelial cells are collected from the oral cavity of the patient andused for culture.

The oral mucosa has high proliferation potency. In the oral mucosa,generally, since the injury is cured after the operation byadministering internal antimicrobial drug and carrying Out disinfectionwith Isodine, and the like, for several days, the invasion with respectto the patient who was subjected to collection of mucosa seems to belight.

On the other hand, as the second cells, another individual's (alto)corneal epithelial cells can be preferably used. As such cornealepithelial cells, cells from donor's eyeball free from infection areavailable from, for example, eye bank (Northwest eye bank, etc.). Thecells that can be used as the second cell are not limited to the cornealepithelial cells. Conjunctival epithelial cells, amnion epithelial cell,and the like, may be used. However, when the corneal epithelial cellsconstituting the corneal epithelium in a living organism or theconjunctival epithelial cells existing in the vicinity thereof areemployed, it is thought that a sheet-shaped composition capable ofreproducing the property of the corneal epithelium more excellently. Asa result of the present inventor's investigation, when the cornealepithelial cells are used as the second cell, it was confirmed that acell layer similar to the corneal epithelium was constructed. This factsupports the above-mentioned prediction and supports that the cornealepithelial cells are particularly preferable for the second cells. Onthe other hand, it was confirmed that when the amnion epithelial cellswere used as the second cell, a cell layer capable of excellentlyreproducing the properties required for the cornea was formed. This factshows that the amnion epithelial cells can be also preferably used asthe second cells.

Autologous cells can be used as the second cells. However, when otherindividuals' cells are used, the cells can be obtained more easily. Forexample, even when a sheet-shaped composition for the treatment of apatient with bilateral eye disease is produced, the corneal epithelialcells as the second cells are available.

The separately prepared first cells and the second cells (hereinafter,also referred to as “the first cells, and the like”) are seeded onamnion and cultured. In general, the first cells and the second cells,which are prepared in a form of a cell suspending solution, are drippedon amnion and cultured.

Typically, the seeding of the first cells and the seeding of the secondcells are carried out simultaneously (herein, “simultaneously” includesnot only a case where the seeding is carried out literallysimultaneously but also a case where the first seeding is carried outand then the second seeding is carried out without substantial timeinterval). The first and second cells may be seeded at different timing.For example, the second cells may be seeded several minutes to severalhours after the first cells are seeded. Such a time lag in seedingenables, for example, to construct non-uniform cell layer such as celllayer having those regions which are rich in cells derived from thefirst cells are localized.

The ratio of the first cells and the second cells to be seeded is notparticularly limited. Typically, substantially the same number of thefirst and second cells are seeded. In an experiment in which the oralmucosal epithelial cells were used as the first cells and the cornealepithelial cells were used as the second cells, the ratio of the numberof the first cells: second cells were changed to 3:7, 5:5, and 7:3 andcomparison was carried out. As a result, no difference in terms of thecell proliferation and stratification were clearly observed among them(data not shown).

When the first and second cells are cultured on amnion, these cells areproliferated and a cell layer is formed (in this process, at least apart of the cells are thought to be differentiated). After the formationof a cell layer, a step of bringing the surface layer of the cell layerinto contact with the air is carried out. This step is also referred toas air lifting in this specification. This step is carried out fordifferentiation of cells forming a cell layer and inducing the barrierfunction.

This step can be carried out by lowering the surface of the culturemedium by temporarily removing a part of the culture medium by using adropper, a pipette, and the like, thereby temporarily exposing theoutermost layer of the cell layer to the outside of the culture medium.Alternatively, this step can be carried out by lifting up the cell layertogether with the amnion, thereby temporarily exposing the outermostlayer from the culture medium surface. Furthermore, by using the tubeetc., the air may be fed into the culture medium so as to bring theuppermost layer of the cell layer into contact with the air. From theviewpoint of the ease in operation, it is preferable that by loweringthe surface of the culture medium, thereby exposing the outermost layerof the cell layer to the outside.

The duration for carrying out this step, that is, the period of timewhen the uppermost layer of the cell layer is brought into contact withthe air differs depending upon the state of the cells, cultureconditions, and the like, but the duration may be, for example, threedays to two weeks, preferably within a week, and further preferablywithin three days.

According to the above-mentioned method of the present invention, on theamnion, a corneal epithelium-like cell layer, in which the first cellsand the second cells are stratified, is formed. The thus obtainedsheet-shaped composition together with the amnion used as a substrate ofthe first cells and the second cells can be used as a transplantationmaterial (substitute for the corneal epithelium) for patients withinjured or defective cornea. In this case, the sheet-shaped compositionis transplanted to the corneal epithelium defective part so that theamnion is located to the side of the eyeball.

In one embodiment of the present invention, cells of biological originare cultured in the presence of support cells. The support cell is alsoreferred to as a feeder cell and supplies a culture medium with a growthfactor, etc. When the cells of biological origin are cultured in thecoexistence of the support cells, the proliferation efficiency of cellsis improved. As the support cell, for example, a 3T3 cell (Swiss mouse3T3 cell, mouse NIH3T3 cell, 3T3J2 cell, etc.) and the like, may beused. Among them, it is preferable to use a mouse NIH3T3 cell as asupport cell from the viewpoint of proliferation efficiency, ease inhandling, etc.

It is preferable that the support cells are inactivated by usingmitomycin C, etc. This is advantageous because the inhibition of theproliferation of the cells of biological origin due to the proliferationof the support cells themselves is prevented, and the proliferationefficiency of the cells of biological origin is enhanced. Suchinactivation can be carried out by a radiation treatment, and the like.

The cell density of the support cells may be, for example, about 1×10²cells/cm² or more, preferably in the range from about 1×10² cells/cm² toabout 1×10′ cells/cm ², and further preferably in the range from about1×10³ cells/cm² to about 1×10³ cell's/cm². As to the ratio with respectto the number of the first cells and the second cells, culture may becarried out under the conditions in which the number of the supportcells to be used may be, for example, 1/10³ times to 1×10² times, andpreferably 1/10² times to 1 time as the total number of cells orbiological origin. When the number of the support cells is small, theproliferation rate of the first cells and the like is lowered; and whenit is too small, excellent proliferation and stratification of cells ofbiological origin cannot be obtained. On the other hand, it is notpreferable that the number of the support cells is too large, becausethe proliferation rate of the oral mucosal epithelial cells is lowered.

When the cells of biological origin are cultured in the coexistence ofsupport cells, it is preferable that an isolation membrane having a poresize to which the support cells cannot pass is provided between thesupport cells and the amnion. The use of the isolation membrane makes itpossible to prevent the support cells from entering the side of theamnion (i.e. the side of living organism cells) at the time ofculturing. As a result, the support cells may not be mixed in thefinally obtained sheet-shaped composition. This means that asheet-shaped composition being free from problem of immunologicalrejection by the support cells can be constructed. Clinically, this isextremely significant.

As the isolation membrane, an isolation membrane having a pore sizethrough which the support cells cannot pass can be used by appropriatelyselecting the known membrane: For example, a polycarbonate membranehaving a pore size of about 0.4 μm to 3.0 μm can be used. A material ofthe isolation membrane is not particularly limited. In addition topolycarbonate, polyester and the like may be used. Such isolationmembranes are on the market and easily available.

Example of the culture method using an isolation membrane include thefollowing method. Firstly, inactivated support cells are seeded andcultured on a container such as a dish (a first container), therebyforming a layer of support cells on the surface of the container. Next,a second container, which has a bottom face made of an isolationmembrane, is set in the first container so that the bottom face of thesecond container is located in a culture medium. Then, the amnion isformed on the bottom face, that is, on the isolation membrane. Then, onthe collagen layer, the cells of biological origin are seeded andcultured.

In one example, on bottom surface of the second container, amnion ispreviously formed (for example, on the bottom surface of the secondcontainer, the amnion from which an epithelium has been removed isplaced. In this state, drying process is carried out). This secondcontainer is set in the first container in which support cells areseeded, and then on the collagen layer, the first cells and the like maybe seeded and cultured.

The culture medium used for culturing the cells of biological origin isnot particularly limited as long as the cells can be proliferated andstratified. For example, a culture medium, in which DMEM (Dulbecco'smodified Eagle's medium) that is generally used for growing epithelialcells and Ham's F12 medium are mixed with each other at thepredetermined ratio, and FBS, growth factor, antibiotics, and the likeare added, may be used. Specific examples include a mixed culture mediumof DMEM and Ham's F12 medium (mixing volume ratio of 1:1) to which FBS(10%), insulin (5 mg/ml), cholera toxin (0.1 nM), epithelial cell growthfactor (EGF) (10 ng/ml) and penicillin-streptomycin (50 IU/ml) areadded. Furthermore, a mixed culture medium of DMEM and Ham's F12 mediumto which triiodothyronine (for example, 2 nM), glutamine (for example, 4mM), transferrin (for example, 5 mg/ml), adenine (for example, 0.18 mM),and/or hydrocortisone (for example, 0.4 mg/ml) are further added, may beused.

The cells of biological origin may be cultured in the absence ofxenogeneic cells. The “the absence of xenogeneic cells” in the presentinvention means that cells of animals different from the cells ofbiological origin are not used as a condition for culturing the cells ofbiological origin. Specifically, when human cells (for example, humanskin epidermal cells or human corneal epithelial cells) are used, thecondition means that cells from the animal species other than human, forexample, a mouse, a rat, or the like, are not present (do not coexist).When cells are cultured in such a condition, xenogeneic components(including xenogeneic cells themselves) may not be contaminated in thefinally obtained transplantation material (that is, sheet-shapedcomposition).

The culture medium used for culturing cells of biological origin is notparticularly limited as long as it allows the cells to proliferate. Forexample, an MCDB153 medium (NISSUI PHARMACEUTICAL CO., LTD.), EpiLife™(Cascade), and media produced by modifying the amino acid composition,etc. of these media, a culture medium mixing DMEM (Dulbecco's modifiedEagle's medium) and Ham's F12 medium, which are usually used for growingepithelial cells, at a predetermined ratio can be used. In particular,in the present invention, it is preferable that a culture medium thatdoes not contain serum and xenogeneic proteins is used. On the otherhand, a culture medium containing growth factor, antibiotics, and thelike may be used. However, it is preferable to use a culture medium thatdoes not contain any serum. That is to say, it is preferable that serumfree culture is employed as a culture method in the present invention.This is advantageous because problem such as immunological rejection dueto the contamination of components derived from the serum can beavoided. Note here that culture may be carried out in a culture mediumcontaining serum, in this case, however, it is preferable to useallogeneic serum (when cells of human origin is used, serum of humanorigin) or to use autologous serum. Needless to say, if possible, it ispreferable to use autologous serum capable of avoiding causing theimmunorejection.

The culture conditions may be changed in the course of culture for thepurpose of excellently proliferating cells of biological origin.

As a result of the culturing step, cells of biological originproliferate on the amnion. When the surface layer of the thus obtainedcell layer is required to be keratinized (for example, a case whereepidermal cells are used so as to form a skin epidermal sheet or a casewhere corneal epithelial cells are used so as to form a cornealepithelial sheet), the above-mentioned Air-lifting may be carried out.

The cells of biological origin are seeded on the amnion so that, forexample, the cell density becomes about 1×10′ cells/cm² or more,preferably in the range from about ×10′ cells/cm² to about 1×10⁷cells/cm², and further preferably in the range from about 1×10⁴cells/cm² to about 1×10⁶ cells/cm².

In one preferable embodiment, amnion is placed on a collagen matrixcontaining human fibroblasts, which has been previously prepared, andthen the cells of biological origin are seeded on the amnion andcultured. That is to say, in this embodiment, a step of culturing humanfibroblasts in a collagen gel (the step B) and a step of placing amnionon the collagen gel, followed by seeding or placing cells of biologicalorigin on the amnion (the step C) are carried out. The sheet-shapedcomposition that has been produced by this procedure has come to containthe cells of biological origin proliferated on the amnion placed on thecollagen gel containing human fibroblasts. The sheet-shaped compositionof this embodiment can be also used as a transplantation material afterthe collagen matrix is removed. Alternatively, the sheet-shapedcomposition of this embodiment can be also used as a transplantationmaterial in a state in which it includes the collagen matrix.

“Collagen gel” functions as a culture substrate of human fibroblasts.The types of collagens as a material of the collagen gel are notparticularly limited, and type I collagen, type III collagen, and typeIV collagen, and the like, can be used. A plurality of collagens can beused in combination thereof. Such collagens can be extracted andpurified from the connective tissue of the skin and cartilage, etc. ofanimals such as pig, bovine, sheep, etc., by an acid solubilizationmethod, an alkali solubilization method, and an oxygen solubilizationmethod, and the like. For the purpose of deteriorating the antigenicity,it is preferable to use so-called atherocollagen obtained by removingtelopeptide by a treatment with the use of catabolic enzyme such aspepsin, trypsin, etc. As materials of the collagen gel, a collagenderived from amnion, particularly derived from human amnion may be used.Herein, the collagen layer is “derived from amnion” means that thecollagen gel is obtained by using amnion as a starting material.

The origin of the human fibroblasts contained in the collagen gel is notparticularly limited and it may be derived from any tissue as long asthe tissue produces collagen. Human fibroblasts prepared from, forexample, skin tissue, oral mucosa tissue, and the like, can be used.

A specific example of the method of producing a collagen matrix isshown. Firstly, human fibroblasts are prepared by the followingprocedure. The skin is collected, and then dermis is peeled off from theskin. The dermis is cut in strips and is brought into close contact witha dish coated with type I collagen. After static culture, humanfibroblasts migrated from the dermis strip are subcultured. Cells arepeeled off from the bottom surface of the dish and a cell suspendingsolution is prepared. The cell suspending solution is seeded on a cellculture dish. Appropriately, cells are cryopreserved (for example,stored in liquid nitrogen).

Meanwhile, a neutralized collagen solution is prepared by using type Icollagen (see the below-mentioned Example). This. is added in a culturecontainer (for example, a culture insert) and stood still for tenminutes at room temperature so as to be gelled. Next, human fibroblastsin a logarithmic growth phase, which has been cultured by theabove-mentioned method in advance, are mixed with this gel and gelledagain. Thereafter, static culture is carried out. A collagen matrixcontaining human fibroblasts can be obtained by the above-mentionedprocedure. This inventiveness allows the collagen matrix to havenecessary strength and to have amnion layer or cells of biologicalorigin to be mounted thereon, which makes a base of the presentinvention. A separately prepared amnion can be placed on (brought intocontact with) the collagen matrix. Thereafter, cells are seeded andcultured in accordance with the above-mentioned procedure.

If the process of attaching adhesive components (fibrinogen, etc.) tothe amniotic surface is performed, the formation of cell layer precedesthe attachment of adhesive components. In order words, in thisembodiment, cell layer is formed on the amnion, and then fibrinogen andany other adhesive components are attached to the amniotic surface(surface where no cell layer is formed).

EXAMPLE 1 1. Trehalose Treatment/Preparation of Lyophilized Amnion 1-1.Harvesting of Amnion

A sufficient informed consent was obtained in advance from a pregnantwoman who had no systemic complications but was undergoing a caesareanoperation, in the presence of an obstetrician. An amnion was obtainedfrom the woman during the caesarean operation in operation room. Theoperation was performed with cleanness being ensured, and with adedicated garment worn after scrubbing according the surgical procedure.Before delivery, a clean vat for harvesting amnion and physiologicalsaline for washing were prepared. After delivery, placental tissue wastransferred to the vat and amniotic tissue was manually detached fromthe placenta. Any adhesion between the amnion and placenta was cut offusing a scissor.

1-2. Treatment of Amnion

Treatment of amnion was performed in the order of (1) washing, (2)trimming, and (3) storage. In any of these steps, the procedure waspreferably performed under a draft, the container and instruments thatwere used had been previously sterilized, and disposable type of dish,etc were used. Blood components attached to the amnion obtained werewashed off with physiological saline, and an additional sufficientamount of physiological saline (0.005% ofloxacin added) was used to washfurther. Subsequently, phosphate buffered saline (PBS) added withpenicillin-streptomycin (50 IU) was used to wash three times in total.Then, the amnion was transferred to the dish, and cut into pieces ofabout 4×3 cm with scissors. After the cutting, amnions in a goodcondition were selected based on the shape and thickness.

1-3. Storage of Amnions 2 cc sterilized cryotubes received preservationsolution 1 cc each, into which one piece each of harvested and washedamnion was put. The cryotubes were labeled and stored in at −−0° C. in adeep freezer. As the preservation solution, solution of 50% sterilizedglycerol in DMEM (Dulbecco'S Modofied Eagle Medium: GIBCOBRL) was used.The duration for use of the preserved amnion was determined to be threemonths, and at the end of the duration, the cryotubes were burned anddiscarded. The described storage process can be done without, and thefollowing epithelium treatment may be performed.

1-4. Treatment of Amniotic Epithelium

The amnion which had been stored at −80° C. was thawed in a roomtemperature, and was washed twice with phosphate buffered solution (PBS)added with penicillin-streptomycin (50 IU). The washed amnion wasimmersed in 0.02% EDTA solution (Nacalai tesque) (100 mm dish), andreacted in a CO2 incubator at 37° C. for one hour. After the reaction,the amnion was washed twice with a sufficient amount of PBS, and had itsepithelium manually peeled off (removed) using cell scraper (Nunc, USA)under stereoscopic microscope. Complete removal by peeling-off ofadditional amniotic epithelium by the process was ensured by inspectionunder light microscope and electron microscope (electron microscopescanning).

1-5. Treatment by Trehalose/Production of Lyophilized Amnion

The amnion with an epithelium removed is immersed in 10% (w/v) trehalosesolution at 37° C. for two hours. The trehalose solution was prepared bydiluting trehalose (Torehainochi, H plus V Lifescience, Hayashibara)with distilled water. pH of the solution was maintained in the rage of 7through 10. The amnion was clamped with a pair of sterilized plasticframes, and secured -with clip. Each set of the frame was transferredinto a deep freezer at −80° C., and, upon determination of freezing ofthe amnion, lyophilization process (−110° C., about one hour) wasperformed using a vacuum lyophilizer (Yamato, NEOCOOL). Conditions wereset according to the instruction from the manufacturer such thatsufficiently desiccated products can be obtained. The amnion after thelyophilization process was released from the frame, transferred into atwo-layered bag made of polyamidenylon on the outside and polyethyleneon the inside, and packed in vacuum using a vacuum packer for home use(Framenova, Magicpack). The amnion packed in vacuum was irradiated withγ-ray (about 25 kGy) to sterilize it. The sterilized amnion was storedin the vacuum package at a normal temperature until immediately beforeuse. The state immediately after the lyophilization process was stillmaintained even 12 months after start of the storage. Furtherexperiments were performed using the desiccated amnion which was storedat a normal temperature for one month.

2. Treatment with Trehalose/Evaluation of Physical Properties ofLyophilized Amnion

Trehalose-treated and lyophilized amnion (also referred to astrehalose-treated FD-AM) prepared in the above procedure was immersed inPBS at a room temperature until its sufficient recovery, and itsphysical properties evaluated. Tested items and method is as follows.The material used for the test was five pieces of trehalose-treated andlyophilized amnion which had been separately prepared. An average of themeasurements obtained was used for the evaluation. Additionally, thoseamnion (raw amnion) which had not underwent none of epithelialtreatment, trehalose-treatment and lyophilization and those amnion whichhad been prepared through similar procedures except the trehalosetreatment were prepared, and served as a standard (control forcomparison) in the evaluation of physical properties.

(1) (Thickness)

Measurement of the thickness was performed using a Double ScanHigh-accuracy laser meter (LT-9010M) from Keyence.

(2) (Transparency)

Tubidimeter (NDH2000) from Nihon-Denshoku was used to measure the hazefor use in evaluation of transparency. Haze is calculated according tothe following equation:

Haze=Diffused Transmission Factor (DF)/Total Light Transmittance (TT)

(3) Tensile Strength

Tensile strength meter (Tensilon RTC-1210A) from A&D was used formeasurement of tensile strength.

(4) Flexibility

Microscope for microsurgery from Karizeiss was used for measuringflexibility by macroscopically detecting presence of wrinkles. Thenumber of wrinkles on the entire eye ball surface was counted by threepersons, and their average was used for evaluating flexibility.

The test results are shown in FIG. 3 through 6. As shown in FIG. 3,trehalose-treated and lyophilized amnion is found to be thicker thanlyophilized amnion. This is thought to be due to a higher waterretention capacity afforded by the trehalose treatment. Removal ofepithelium is reflected upon the significantly reduced thickness of thetrehalose-treated and lyophilized amnion compared to raw amnion (AM).

It is also noted that, as shown in FIG. 4, trehalose-treated andlyophilized amnion has a higher transparency than lyophilized amnion.Surprisingly, it was revealed that trehalose treatment enhancestransparency. The poorer transparency or the raw amnion compared to thetrehalose-treated and lyophilized amnion is due to the presence ofepithelium.

On the other hand, as shown in FIG. 5, trehalose-treated and lyophilizedamnion has a higher tensile strength than lyophilized amnion.Surprisingly, the trehalose-treated and lyophilized amnion has a higherstrength even compared amnion comprising an epithelium (raw amnion).Thus, it was revealed that trehalose treatment is extremely effective inenhancing the strength of amnion.

Further, as shown in FIG. 6, the flexibility of trehalose-treated andlyophilized amnion is far from that of lyophilized amnion, but isequivalent to that of raw amnion. Thus, it was revealed that trehalosetreatment is extremely effective in enhancing flexibility of amnion.

3. Evaluation of Biocompatibility of Trehalose-treated and LyophilizedAmnion

Materials that are transplanted into an organism are required to have ahigh biocompatibility. Therefore, the biocompatibility oftrehalose-treated and lyophilized amnion was evaluated by the followingprocedure.

6 week old Japanese rabbit received an incision with a scalpel on itseye surface into parenchymal layer of cornea. Subsequently, anappropriately sized trehalose-treated and lyophilized amnion wasinserted into the incision of parenchymal layer. After the transplant,the state of the eye surface was monitored for a period of time. Thestates of the eye surface immediately after and one month post thetransplant are shown in FIG. 7. At one month after the transplant, noangiogenesis occurring from surrounding area as well as inflammationreaction were recognized. The transparency of the eye surface wasgreatly enhanced compared to that immediately after the transplant.Thus, it was determined that the biocompatibility of trehalose-treatedand lyophilized amnion is equivalent to that of untreated amnion.

In order to examine the biocompatibility in greater details, a part ofthe cornea including the transplanted portion was subjected to HEstaining at one month post the transplant. Image of the HE staining isshown in FIG. 8. The graft (i.e. trehalose-treated and lyophilizedamnion) is indicated with an arrow. As clearly shown in FIG. 8, none ofabnormal differentiation, intracorneal angiogenesis, edema, and cellularinfiltration is recognized in the epithelium. Consequently, it wasdemonstrated that trehalose-treated and lyophilized amnion has anextremely low antigenicity, and is therefore superior inbiocompatibility.

4. Production of Cultured Corneal Epithelial Sheet UsingTrehalose-Treated and Lyophilized Amnion 4-1. Recovery of CornealEpithelial Cells

Corneal harvested from 6 week old Japanese white rabbit was immersed inDMEM containing 10% fetal bovine serum (FBS), and had its conjunctiva,corneal endothelium and other unnecessary tissue excised. Tissue wasthen washed with phosphate buffered solution (PBS) and immersed inphosphate buffered solution (PBS) containing 1.2 U/ml of dispase(Nacalai tesque) at 37° C. for one hour. The tissue after the treatmentwas taken out, and immersed in 0.02% EDTA at a room temperature for twominutes, and then in phosphate buffered solution at a room temperaturefor two minutes to arrest the dispase activity. Corneal epithelial cellswere peeled off in DMEM containing 10% fetal bovine serum (FBS), andsubjected to centrifugation to concentrate and recover the cornealepithelial cells.

4-2. Preparation of Cocultured Cells

NIH-3T3 cells (hereinafter, referred to simply as “3T3 cells”) were usedas cocultured cells (supporting cells). 3T3 cells previously cultured toconfluent in 75F flask (BD Falcon) were immersed in 0.05% mitomycin Csolution for two hours to suppress the proliferative activity of 3T3.Subsequently, the cells were washed several times with phosphatebuffered solution (PBS) to remove mitomycin C. The cells were thentreated with 0.05% trypsin-EDTA solution, and pippeted to provide a cellsuspension (3T3 cell suspension).

4-3. Formation of Cell Layer

Trehalose-treated and lyophilized amnion obtained in 1 was immersed at aroom temperature in PBS until sufficient recovery. The amnion preparedin this manner was used as substrate for co-culturing corneal epithelialcells and 3T3 cells according to the following procedure. As cultureinstruments, 6 well-culture dish (Corning, N.Y.) and culture insert(culture insert container) (made of polycarbonate, average pore size 3.0μm, Corning, N.Y.) were used.

The culture dish was first seeded with 3T3 cell suspension to a celldensity of about 1×10° cell/cm² and incubated at 37° C. under 5% CO₂.Meanwhile, amnion was attached on the culture insert, with its side ofbasal membrane (where the epithelium was present) facing upward, anddried at a room temperature for ten minutes. Then, the culture inserthaving the amnion attached was seeded with suspension of cornealepithelial cell to a cell density of about 1×10⁵ cell/cm².

Following the above procedure, the culture insert was placed inside theculture dish, as shown in FIGS. 9, and 3T3 cells and corneal epithelialcells were cultured on the same culture media. FIG. 9 is a sectionalschematically showing the state during the culture. Culture insert 12stands still inside culture dish 11, on the bottom of which is formed3T3 cell layer 15. On the bottom of culture insert 12 is amnion 13standing still, on which corneal epithelial cells 14 are cultured.Numeral 16 represents culture media.

The media used was DMEM/ham F12 mixed media (mixed volume ratio 1:1)added with 10% FBS, insulin (5 mg/ml), cholera toxin (0.1 nM),penicillin streptomycin (50 IU/ml), human recombinant epithelial cellgrowth factor (EGF) (10 ng/ml).

3 week cultivation was performed in the above media (Submerge). Then, inorder to induce differentiation of mucosal epithelium, so called“air-lifting” method was used to continue the cultivation for furtherone week. In the air-lifting method, the surface level of the media isbrought to level with the corneal epithelial cell-derived cell layerformed on the amnion, while exposing the surface of the cell layer tothe air. During the submerge process, the media was exchanged on everyother day, and, after the air-lifting process, was exchanged daily toperform the cultivation. As result, cell layer was formed on the amnion.

5. Verification of Histological Properties of Cultured CornealEpithelial Sheet.

At 2 days of cultivation after the air-lifting, cell layer similar tocorneal epithelium was formed (FIG. 10, right). It can be seen in thecell layer that cells are 5-7 layered in the same manner in normalcorneal epithelium. Amniotic side of the cell layer had cells similar tobasal cells in a relatively columnar shape. Those cells in outermostlayer were flat and had nuclei while not keratinized on its surfaceunlike skin. Thus, it was ascertained that cell layer similar to cornealepithelium (corneal epithelium-like layer) was formed on the amnion. Onthe other hand, when lyophilized amnion without trehalose treatment(lyophilized amnion) was used as substrate to similarly culture thecorneal epithelial cells, formation of cell layer occurs only in alimited fashion, resulting in 1-2 layers (FIG. 10 left). Therefore, itcan be determined that trehalose-treated and lyophilized amnion canexerts it function to facilitate a normal differentiation of cornea.

Subsequently, immuno-staining was performed to further investigate thehistological properties of the cell layer. First, the cell layerobtained was cut together with the amnion into an appropriate size,frozen-embedded in OCT compound, and then sliced in cryostat to produceslide sections. The immuno-staining was targeted to keratin,representative cytoskeletal proteins. In particular, expressions ofcornea-specific keratin 3, epidermis-specific keratin 10, andconjunctiva-specific keratin 13 were examined according to the followingmethod. Slide section was washed with phosphate buffered solution (PBS)and blocked with 1% fetal bovine serum (FBS) to inhibit non-specificantibody reaction. Then, antibodies (first antibody) specific for eachof the keratins were reacted at a room temperature for one hour. Afterthe reaction, washing was performed in PBS containing Triton-X for 15minutes three times, and fluorescence-labeled antibodies (secondantibody) were reacted at a room temperature for one hour. After thereaction, washing was performed for 15 minutes three times in phosphatebuffer solution (PBS), and, upon mounting, tissues were visualized underconfocal microscope.

The antibody reactions for each of the listed keratins in the cell layerwere as follows. First, there were no staining for epidermis-specifickeratin 10 and conjunctiva-specific keratin 13 evident (FIG. 11, lower).On the other hand, staining for cornea-specific keratin 3 wasextensively evident (FIG. 11 upper right). The staining for keratin 3was intense in upper portion of the cell layer. Based on these results,it was demonstrated that epithelial cell layer similar to normal cornealepithelium was formed.

6. Transplant Experiment Using Cultured Corneal Epithelium Sheet

The sheet produced in 4 which had corneal epithelium-like cell layerformed there (Cultured corneal epithelium sheet) was used to perform thefollowing transplant experiment.

First, the rabbit from which corneal epithelial cells had been harvestedreceived ablation with crescent knife to thoroughly remove the areaspanning 4 mm from its limbus to corneal and conjunctival epithelium ina depth of 100 μm. Since this ablation eliminates epithelial cellsincluding corneal epithelial stem cells, it is believed that artificialeye surface stem cell exhaustion is reproduced. Subsequently, culturedcorneal epithelium sheet was transplanted to a region slightly inwardfrom the limbus. 10-nylon thread was used for the transplant to performthe suture with surrounding tissues. After the transplant, therapeuticcontact lens was sutured on the graft. Upon completion of the surgery,antibiotics and steroid eye lotion were applied twice a day. The eyesurface after the transplant had a similar transparency to that ofcultured corneal epithelium sheet prior to the transplant.

The eye surface that had undergone the transplant was visualized twodays and 14 days after the transplant. At the same time, fluoresceinstaining test was performed by applying fluorescein test papercontaining moisture such as antibiotic eye drop directly onto eyesurface, making the subject blink several times, and visualizing thefluorescein staining of the eye surface. If corneal epithelium remains,its intercellular adhesive structure prevents fluorescein dye frominfiltrating, thereby producing no staining by fluorescein.

At two days after the transplant, the eye surface retained itstransparency (FIG. 12, upper left). In addition, it was revealed byfluorescein staining that cultured corneal sheet remained on the eyesurface without any defect (FIG. 12, lower left). On the other hand,based on the observation that transplanted cultured corneal epitheliumsheet shows no fluorescein staining, it was demonstrated that culturedcorneal epithelium sheet had a similar barrier function to that ofcorneal epithelium. Further, it was noted that fluorescein staining wasevident all around the periphery of the transplanted cultured cornealepithelium sheet, thus demonstrating that those tissues which waspresent on the transplanted was not due to contamination by surroundingresidual conjunctival epithelium.

Since cells in corneal epithelium typically bind to each other in atight adhesive structure, fluorescein dye does not infiltrate thesurface. Specifically, no staining is shown in fluorescein stainingtest. In contrast, when those cells had their adhesion loosened or hadtheir barrier function disrupted due to detachment of cells themselves,infiltration of fluorescein dye is allowed to stain the tissue.Accordingly, by examining any staining by fluorescein dye, it can bedetermined whether or not transplanted cultured corneal epithelium sheethas a similar barrier function to that of corneal epithelium.

On the other hand, it was observed that at 14 days after transplant, thecultured corneal epithelial sheet still remained on the eye surface, andadditionally, extended toward periphery compared the state at 2 daysafter the transplant, covering the entire eye surface (FIG. 12, upperright). Also, it was observed that the eye surface itself showed nofluorescein staining, indicating that the cultured corneal epitheliumsheet retained its barrier function (FIG. 12 lower right). Transparencyhad no changes from that at 2 days after the transplant, and wasretained at a high level (FIG. 12 upper right).

Based on the above results, it was demonstrated that cultured cornealepithelium sheet obtained by using trehalose-treated and lyophilizedamnion as a substrate is provided with a favorable take to eye surface,which is maintained for an . extended time period. Moreover, its wasobserved that the sheet extends to periphery after transplant to exert abarrier function required as corneal epithelium for an extended timeperiod, while maintaining a high transparency. Specifically, it wasobserved that the cultured corneal epithelium sheet obtained accordingto the above method serves favorably as a substitute for cornealepithelium, and can be favorably used as a graft for reconstructing eyesurface, for example in the event where any damage or defect has beencaused on cornea.

7. Evaluation of Histological Properties of Transplanted CulturedCorneal Epithelium Sheet

The cultured corneal epithelium sheet was removed two weeks after thetransplant for inspection of its histological properties. FIG. 13 showin upper left image HE staining image of the cultured corneal epitheliumsheet. Cell layer where cells are regularly arranged, similarly to thatof normal corneal epithelium, can be found on trehalose-treated andlyophilized amnion (TH-AM, marked with **). In that image, the uppercell layer has many more flat cells, and maintains a structure extremelysimilar to that of corneal epithelium.

Results of staining test for each of the keratins are shown in upperright image and lower image of FIG. 13. Generally similar staining tothose of cultured corneal epithelium, sheet prior to the transplant wasshown. Specifically, staining for the epidermis-specific keratin 10 andconjunctiva-specific keratin 13 were not evident (FIG. 13, lower), whilestaining for only corneal-specific keratin 3 was evident for the entirecell layer (FIG. 13, upper right). Thus, it was determined that thecultured corneal epithelium sheet, even after the transplant, maintainedthe corneal-specific keratin. This result supports from the histologicalview point that cultured corneal epithelial sheet exerts a similarfunction to corneal epithelium for an extended period of time.

8. Immuno-staining for Basal Membrane and Stratum Compactum Components

In order for amnion to favorably act as a substrate for cell culture, itis believed that the basal membrane and stratum compactum preferablyhave retained their innate structures. Whether or not the basal membraneand stratum compactum have retained their innate structures can beevaluated by examining the presence or absence (whether or not they areretained) of components characteristic of each of these. Thus, thefollowing immuno-staining method was used to examine whether or not thebasal membrane and stratum compactum components are retained intrehalose-treated and lyophilized amnion. In this method, amnion thathad undergone none of epithelium process, trehalose treatment processand lyophilization process (raw amnion) and amnion that had beenprepared in the same manner as trehalose-treated and lyophilized amnionexcept that it had not undergone trehalose treatment (lyophilizedamnion) were compared.

First, each amnion was cut into a size of 1.5×1.5 cm, embedded in OCTcompound, and frozen at −80° C. to provide frozen preparations. Thesepreparations in the frozen state were sliced into a thickness of 8 μm inthe vertical direction to amniotic surface in cryostat (CM1900 Leica),and mounted on glass slide to provide as frozen section. These sectionswere used in the immuno-staining according to the following procedure.

1. Acetone fixation, 5 min., 2. Washing in PBS, 30 min., 3. Blockingwith PBS/3% BSA 15 min., 4. First antibody, one hour, 5. Washing in PBS,30 min. 6. Blocking with PBS/3% BSA, 15 min., T. Second antibody, onehour, 8. Washing in PBS, 30 min., and 9. Mounting.

Samples after mounting were visualized under fluorescence microscope(Leica DMIRB).

Antibodies that were used are as follows. Dosages were determinedaccording to the instruction from the manufacturers.

Collagen I (Collagen I): LSL LB-1190, Collagen III (Collagen III): LSLLB1300, Collagen IV (Collagen IV): LSL LB-1407, CollagenV (Collagen V):LSL LB1581, Collagen VII (Collagen VII): Chemicon MAB1345, Laminin 5(Laminin-5): Chemicon MAB19562, Fibronectin (Fibronectin): LSL LB-1021.

Amniotic basal membrane layer has expressions of Collagen IV, VII andLaminin 5, while stratum compactum layer has expressions of Collagen I,III, V, and Fibronectin. Therefore, immuno-staining using antibodies foreach of these allows for visualization of any amniotic basal membraneand stratum compactum that were retained. In addition, since PI stainingis also performed in the instant experiment, the presence or absence ofamniotic epithelial cells can be simultaneously determined.

Results of the immuno-staining are shown in FIG. 14. Images in column Aare staining images of raw amnions, those in column B are stainingimages of lyophilized amnions, and those in column C are staining imagesof trehalose-treated and lyophilized amnions. The images in each columnare, in the order from the uppermost one to the lowermost one, (1)staining images of Collagen I, (2) staining images of Collagen III, (3)staining images of Collagen IV, (4) staining images of Collagen V, (5)staining images of Collagen VII, (6) staining images of Laminin 5, and(7) staining images of Fibronectin.

The following facts were revealed as result of the staining. First,compared to lyophilized amnion, trehalose-treated and lyophilized amnionhad stronger signals for basal membrane components (Collagen IV,Collagen VII, Laminin 5), showing the staining for basal membranecomponents that is similar to that of raw amnion. This indicates thattrehalose-treated and lyophilized amnion highly maintains its innatestructure of the basal membrane. In a part (Collage IV and Fibronectin)of the staining results, staining was evident on the entire lyophilizedamnion, while trehalose treated and lyophilized amnion had relativelyclear confines of stained region and non-stained region, similarly toraw amnion. This indicates that trehalose treated and lyophilized amnionhighly retains its innate structures basal membrane and staratumcompactum.

As can be apparently shown in the staining result of the stratumcompactum, trehalose-treated and lyophilized amnion has stratumcompactum that is thicker than that of lyophilized amnion. It wasrevealed that, when returned to a moistened state, a favorable swell ofstratum compactum as well as recovery of thickness to a similar level tothat of raw amnion resulted from the trehalose treatment.

Thus, it was thus revealed that trehalose treated and lyophilized amnionhas structures of basal membrane and stratum compactum that are similarto those of raw amnion. Specifically, it was ascertained that trehalosetreatment prevents the structures of basal membrane and stratumcompactum from being damaged during lyophilization, thereby providingamnion with structures of basal membrane and stratum compactum that aresimilar to raw amnion.

9. Review of Method for Removing Epithelium

Amnion was processed according to the procedure shown in FIG. 15 toprepare amnion with its epithelium removed (non-epithelium-containingamnion). Operation method (processing method), operation condition(processing condition), and other details will now be described.

9-1. Harvesting of Amnion

A sufficient informed consent was obtained in advance from a pregnantwoman who had no systemic complications but was undergoing a caesareanoperation, in the presence of an obstetrician. An amnion was obtainedfrom the woman during the caesarean operation in operation room. Theoperation was performed with cleanness being ensured, and with adedicated garment worn after scrubbing according the surgical procedure.Before delivery, a clean vat for harvesting amnion and physiologicalsaline for washing were prepared. After delivery, placental tissue wastransferred to the vat and amniotic tissue was manually detached fromthe placenta. Any firm adhesion between the amnion and placenta was cutoff using a scissor.

9-2. Removal of Blood Components and Detachment of Chorionic Membrane

Blood components attached to the amnion obtained were washed off withphysiological saline, and an additional sufficient amount ofphysiological saline (0.005% ofloxacin added) was used to wash further.Then, chorionic membrane adhered to the amnion was manually removed.

9-3. Securing of Amnion

Amnion was secured according to the method shown in FIG. 2 b. First, theamnion was mounted on a sterilized film made of fluorocarbon resin withepithelial side facing upward. The amnion was then spread wide toeliminate any wrinkle and sag, and a sterilized sheet frame made offluorocarbon resin was mounted on the amnion. Clips or other device wasused to secure the sheet of fluorocarbon resin and the sheet frame offluorocarbon resin, thereby clamping the amnion between the fluorocarbonresin-made sheet and the fluorocarbon resin-made sheet frame (Scuring offrame). An excess portion of the amnion that protruded was cut out.Epithelial side's facing upward was ensured by observation understereoscopic microscope.

9-4. Freeze-Thawing Process

The amnion secured into a frame was transferred in to a deep freezer at−80° C., and left stood for about 30 minutes (Freezing). The amnion wasthen taken out from the deep freezer, transferred into an incubator at37° C., and left stood for about 30 minutes (Thawing). The aboveprocedure was performed another time.

9-5. Tryptic Treatment

Following the freeze-thawing process, the amnion was immersed in trypticsolution (0.02% trypsin, 0.2 mM EDTA-containing phosphate buffersolution) with the amniotic epithelial side facing upward, and leftstood for about 15 minutes (37° C.). The immersion method was as shownin FIG. 16 a. Specifically, amnion 10 secured into a frame was retained,with the fluorocarbon resin-made sheet 4 facing downward, and trypticsolution 5 was added into fluorocarbon resin-made sheet frame 3. Thisresults in immersion of only the epithelial portion of amnion 10 intryptic solution 5. As shown in FIG. 16 b, amnion 10 secured into aframe may be put into container 6 with the epithelial side facingdownward, thereby attaining immersion in tryptic solution 5: In theinstant example, by engaging protrusions on the inside of container 6with fluorocarbon resin-made sheet frame 3′, a desired relative positionbetween liquid surface 5 a of tryptic solution 5 and amnion 10.

9-6. Washing

Following the tryptic treatment, the amnion is removed from thefluorocarbon resin-made sheet and fluorocarbon resin-made sheet frame,transferred into PBS, and washed by shaking therein (140 rpm 15 minutes,twice). This procedure removes tryptic solution and amniotic epithelialcells.

9-7. Lyophilization

The amnion after washing was clamped by a pair of sterilized plasticframes, and secured. Each of the frames was separately transferred intodeep freezer at −80° C., and, upon determination of freezing of theamnion, lyophilization process (−110° C., about an hour) was performedusing vacuum dryer (Yamato, NEOCOOL). Conditions were set according tothe instruction from the manufacturer such that sufficiently desiccatedproducts can be obtained. The amnion after the lyophilization processwas released from the plastic frame, transferred into a two-layered bagmade of polyamidenylon on the outside and polyethylene on the inside,and packed in vacuum using a vacuum packer for home use (Framenova,Magicpack). The amnion packed in vacuum was irradiated with γ-ray (about25 kGy) to sterilize it. The sterilized amnion was stored in the vacuumpackage at a normal temperature until immediately before use. The stateimmediately after the lyophilization process was still maintained even12 months after start of the storage.

9-8. Review of Freeze-Thawing Process and Removal Method of Epitheliumby Tryptic Treatment

The method of removing epithelium as described above was evaluatedaccording to the following procedure. The evaluation experiment usedepithelium-non-containing amnion (trypsin-treated amnion) that isobtained by washing the amnion post tryptic treatment.

(1) HE Staining

Epithelium-non-containing amnion (trypsin-treated amnion) was HE stainedaccording to the following method. Amnion which had not undergoneepithelium removal (raw amnion) and that with its epithelium manuallyremoved (manually treated amnion) were used as compared subjects.

First, each of eithelium-non-containing amnion (trypsin-treated amnion),raw amnion and amnion with its epithelium manually removed (manuallytreated amnion) were cut into a size of 1.5×1.5 cm, embedded in OCTcompound, and frozen at −80° C. to provide frozen preparations: Thesepreparations in the frozen state were sliced into a thickness of 8 μm inthe vertical direction to amniotic surface in cryostat (CM 1900 Leica),and mounted on glass slide to provide as frozen section.

Procedures and conditions for the HE staining were as follows. 1. 10%formaldehyde solution 5 min., 2. Washing in Running Water, 15 min., 3.Hematoxylin solution, 10 sec., 4. Washing in Running Water, 15 min., 5.Eosin solution, 10 min., 6. Washing in Running Water, 15min., 7. 70%ethanol, 10 sec., 8. 90% ethanol, 10 sec., 9. 95% ethanol, 10 sec., 10.100% ethanol, 10 sec., 11. 100% xylene, 10 sec., 12. 100% xylene, 30min. and 13. Mounting.

Samples after mounting were visualized under light microscope (OlympusBX50).

If hematoxyn is used for the staining of amnion, the amniotic epithelialcells are stratum compactum nucleated cells are stained. In contrast, ifeosin is used for staining, stratum compactum is stained. Thus, throughthe HE staining, the presence or absence of detachment of epithelialcells, as well as the presence or absence of any damage to stratumcompactum can be identified.

Results of the HE staining are shown in FIG. 17. Cell layer (epithelium)can be observed in raw amnion. In trypsin-treated amnion, no cell layer(epithelium) can be observed, similarly to manually treated amnion. As aresult, it was determined that epithelium can be removed completely andevenly through the above method: Meanwhile, no damage in the stratumcompactum was found.

(2) Immuno-staining of Basal Membrane and Stratum Compactum Components

In order for amnion to favorably act as a substrate for cell culture, itis believed that the basal membrane and stratum compactum preferablyhave retained their innate structures, in addition to the requirementfor complete removal of epithelium. Whether or not the basal membraneand stratum compactum have retained their innate structures can beevaluated by examining the presence or absence (whether or not they areretained) of components characteristic of each of these. Thus, thefollowing immuno-staining method was used to examine whether or not thebasal membrane and stratum compactum components are retained intrypsin-treated amnion. Similarly to the case of HE staining, amnionwhich had not undergone epithelium removal (raw amnion) and that withits epithelium manually removed (manually treated amnion) were used ascompared subjects. The method for producing the frozen sections ofamnion is similar to that of HE staining. Such sections were used forperforming the immuno-staining according to the following procedure. 1.Acetone fixation, 5 min., 2. Washing in PBS, 30 min., 3. Blocking withPBS/3% BSA, 15 min., 4. First antibody, 1 hour, 5. Washing in PBS, 30min., 6. Blocking with PBS/3% BSA, 15 min., 7. Second antibody, 1 hour,8. Washing in PBS, 30 min. and 9. Mounting.

Samples after mounting were visualized under fluorescence microscope(Leica DMIRB).

Used antibodies were as follows, and dosages were determined accordingto the instruction from the manufacturer.

Collagen I (Collagen 1): LSL′LB-1190, Collagen III (Collagen III): LSLLB1300, Collagen IV (Collagen IV): LSL LB-1407, Collagen V (Collagen V):LSL LB1581, Collagen VII (Collagen VII): Chemicon MAB1345, Laminin 5(Laminin-5): Chemicon MAB19562, Fibronectin (Fibronectin): LSL LB-1021.

Amniotic basal membrane layer has Collagen IV, II and Laminin 5expressed, while stratum compactum has Collagen I, III, V andFibronectin expressed. Therefore, immuno-staining using antibodies foreach of these allows for visualization of any amniotic basal membraneand stratum compactum that were retained. In addition, since PI stainingis also performed in the instant experiment, the presence or absence ofamniotic epithelial cells can be simultaneously determined.

Results of the immuno-staining are shown in FIG. 18 through 21. FIGS. 18and 19 are immuno-staining images of trypsin-treated amnions, FIG. 20 isan immuno-staining image of raw amnion (with epithelium thereon), andFIG. 21 is an immuno-staining image of manually treated amnion. Summaryof the immuno-staining results are shown in FIG. 22. As is apparent fromthese results, trypsin-treated amnion retains it basal membrane andstratum compactum components to a similar degree to those in manuallytreated amnion. Specifically, the above treatment method enables toretain the basal membrane and stratum compactum components equivalentlyto conventional, manual treatment method.

9-9. Summary

It was observed in the above results of experiment that, by processingthe amnion with the freeze-thawing and tryptic treatment in combination,epithelium can be completely removed without substantially damagingamniotic basal membrane and stratum compactum (i.e. with a favorableretention of innate structure).

INDUSTRIAL APPLICABILITY

The sheet-shaped according to the invention can be useful in anextensive field, such as transplant material for reconstructing tissues,and antiadhesive material. Exemplary applied fields for the sheet-shapedcomposition according to the invention are fields of ophthalmology,digestive surgery, gynecology and dermatology.

This invention is not limited in any way by the Mode of Operation andEmbodiments described above. The present invention encompasses variousmodifications that are readily thought of by those who skilled in theart.

Contents of any thesis, Published Patent application and Patent Gazettespecified in the present specification are hereby incorporated byreference for their entirety.

1. A sheet-shaped composition comprising an amnion having its epithelialcell layer removed, said composition lyophilized and trehalose-treated,wherein said composition has an enhanced tensile strength compared toraw amnion and an enhanced flexibility compared to lyophilized amnion.2. The sheet-shaped composition according to claim 1 in a frozen ordesiccated state.
 3. The sheet-shaped composition according to claim 1wherein said composition has an increased clarity compared to asheet-like composition comprising amnion with epithelium removed,lyophilized, which has not been trehalose-treated, and wherein saidincreased clarity is capable of being evaluated by measuring turbidity,calculated according to the equation: Haze=Diffuse Transmission Factor(DF)/Total Light Transmittance.
 4. The sheet-shaped compositionaccording to claim 1, wherein said amnion has basal membrane componentsCollagen IV, Collagen VII, and Laminin 5 that are detected at anequivalent intensity to that in untreated amnion.
 5. The sheet-shapedcomposition according to claim 1, wherein said amnion is a human amnion.6. The sheet-shaped composition according to claim 1, wherein a celllayer consisting of tissue-derived cells is formed on said amnion. 7.The sheet-shaped composition according to claim 6, wherein saidtissue-derived cells are derived from corneal epithelium, conjunctivalepithelium, skin epidermis, follicular epithelium, oral mucosaepithelium, pigment epithelium iris, pigment epithelium retina, airwaymucosa epithelium, or intestinal mucosa.
 8. The sheet-shaped compositionaccording to claim 6, wherein said cell layer is composed of about 5-7layered cells, and has properties similar to those of cornealepithelium.
 9. The sheet-shaped composition according to claim 1, foruse as antiadhesive materials or reconstruction materials for surface oftissues damaged during surgical invasion.
 10. The sheet-shapedcomposition according to claim 1, wherein said amnion has an adhesivecomponent attached on its chorion side surface.
 11. The sheet-shapedcomposition according to claim 12, wherein said adhesive componentcomprises fibrinogen and thrombin.
 12. The sheet-shaped compositionaccording to claim 12, wherein said adhesive component comprisesfibrinogen, thrombin and aprotinin.
 13. The sheet-shaped compositionaccording to claim I, wherein the chorion side surface of the amnion iscovered with bioabsorbable material.
 14. A transplant method using thesheet-shaped composition according to claim 1 as implant material.
 15. Amethod for producing a sheet-shaped composition comprising an amnion,said composition having an enhanced tensile strength compared to rawamnion and an enhanced flexibility compared to lyophilized amnion, saidmethod comprising the steps of: (a) preparing an amnion having itsepithelial cell layer removed; (b) adding trehalose to said amnion; (c)freezing or desiccating said amnion.
 16. The method of claim 15 whereinsaid composition has an increased clarity compared to a sheet-likecomposition comprising amnion with epithelium removed, lyophilized,which has not been trehalose-treated, and wherein said increased clarityis capable of being evaluated by measuring turbidity, calculatedaccording to the equation: Haze=Diffuse Transmission Factor (DF)/TotalLight Transmittance.
 17. The method according to claim 15, furthercomprising the step of: (d) sterilizing said amnion after step (c). 18.The method according to claim 15, wherein step (a) comprises thefollowing steps of: (i) separating an amnion from an organism, (ii)freeze-thawing said amnion, (iii) subjecting said amnion afterfreeze-thawing to tryptic treatment, (iv) washing said amnion aftertryptic treatment.
 19. The method according to claim 18, wherein thefreezing temperature during said freeze-thawing process is from about−20° C. to about −80° C., and the thawing temperature is from about 4°C. to about 50° C.
 20. The method of claim 18, characterized byrepetition of said freeze-thawing process twice or more times.
 21. Themethod of claim 18, characterized by the tryptic treatment beingperformed using a tryptic solution having a tryptic concentration offrom about 0.01% (w/v) to about 0.05% (w/v).
 22. The method according toclaim 21, characterized by the tryptic solution comprising from about0.1 mM to about 0.6 mM of a chelator selected from the group consistingof EDTA, NTA, DTPA, HEDTA, GLDA, and any combination thereof.
 23. Themethod according to claim 18, characterized by the tryptic treatmentbeing performed under the condition such that the tryptic solution iscontacted with only the epithelium side of said amnion.
 24. The methodaccording to claim 15, wherein the following step of forming a celllayer consisting of tissue-derived cells on said amnion is performedafter step (b).